Review Article

Systematic review of hormonal strategies

to improve fertility in rams
Estela Garza-Brenner1 , Fernando Sánchez-Dávila1* , Keyla Mauleón-Tolentino1 ,
Cecilia Carmela Zapata-Campos2 , Carlos Luna-Palomera3 , Javier Hernandez-Melendez4 ,
Marisol Gonzalez-Delgado5 , José Fernando Vázquez-Armijo6 
1
Facultad de Agronomía, Posgrado Conjunto, Universidad Autónoma de Nuevo León, General Escobedo, N.L México
2
Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Tamaulipas, Ciudad Victoria, Tamaulipas, México
3
División Académica de Ciencias Agropecuarias, Universidad Juárez Autónoma de Tabasco, Villahermosa, Tabasco, México
4
Facultad de Ingeniería y Ciencias, Universidad Autónoma de Tamaulipas, Tamaulipas, México
5
Centro de Investigación en Producción Agropecuaria, Universidad Autónoma de Nuevo León, Linares, Nuevo León, México
6
Centro Universitario Temascaltepec, Universidad Autónoma del Estado de México, Temascaltepec, México

How to cite: Garza-Brenner E, Sánchez-Dávila F, Mauleón-Tolentino K, Zapata-Campos CC, Luna-Palomera C,

Hernandez-Melendez J, Gonzalez-Delgado M, Vázquez-Armijo JF. Systematic review of hormonal strategies to
improve fertility in rams. Anim Reprod. 2024;21(2):e20240007. [Link]

Abstract
Reviewing the current state of knowledge on reproductive performance and productive traits in rams has
many advantages. First, the compilation of this information will serve as a literature resource for scientists
conducting research around the world and will contribute to the understanding of the data collected and
interpreted by researchers on the different hormonal strategies used to improve reproductive performance
in rams. Second, it will allow scientists to identify current knowledge gaps and set future research priorities
in ram reproduction. Rams play an important role in the global flock economy, but their reproductive analysis
has been limited in the use of hormonal technologies to increase the productivity of sheep flocks. In this
review, we cite the most important works on six hormones that, in one way or another, modify the
hypothalamus-pituitary-gonadal axis, at different doses, in and out of the reproductive season, breeds,
application methods, among other factors. The overall aim is to increase the reproductive efficiency of rams
in different scenarios and, in some cases, of other species due to the lack of limited information on rams.
Keywords: rams, kisspeptin, eCG, PGF2α, GnRH.

Introduction
According to the FAO, by 2023 (FAO, 2023), the global sheep population will be 1,195 million
head, with 42% concentrated in Asia, followed by 24% in Africa. Only 8.1% of the world's sheep
are concentrated in the Americas (FAO, 2023). Sheep are the major contributors to global
livestock production in virtually all agro-ecological regions (Macías-Cruz et al., 2018a, b). In
many parts of the world, sheep are produced extensively in arid, semi-arid, tropical and
subtropical regions with little or no nutrient supplementation (Sánchez-Dávila et al., 2020;
Stewart et al., 2021). Under grazing conditions, or in less favourable environments, nutrition
plays a determining role in the reproductive process of the ram, but the photoperiod is one of
the main factors that determine the seasonality of the ram (Rosa y Bryant, 2003;
Gündoğan et al., 2003). Hormonal concentrations of LH, FSH, testosterone and prolactin show
seasonal variations, which are mainly regulated mainly by photoperiod. Therefore, a decrease
in photoperiod stimulates the secretion of LH and FSH by the pituitary gland, resulting in
increased testicular activity in testosterone production (Cruz-Espinoza et al., 2021).
Genetic variation in the reproductive potential among populations is large, and genetic
variation within populations is often sufficient for reproductive genetic improvement (Sánchez-
Dávila et al., 2011; Pérez et al., 2020). This reproductive potential can be strongly channelled to

*Corresponding author: fernando_sd3@[Link]

Received: January 16, 2024. Accepted: April 16, 2024.
Financial support: EGB, FSD and KMT was supported in the preparation of this review by the Universidad Autónoma de Nuevo León.
Conflicts of interest: The authors have no conflict of interest to declare.
Copyright © The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Review of the use of hormones in rams

take advantage of the long reproductive season, ovulation rate and age at first lambing that
these breeds present, and linking them to good management, feeding and natural resources
of each region where they are used globally (Sánchez-Dávila et al., 2011.
It is mentioned that at the time of mating, because of seasonality, about 75% of the rams are
optimal for breeding (Pool et al., 2020a), so the implementation of a breeding management program,
with the same attention that is paid to the ewe, should be applied to the males that will be used for
breeding. In this context, the establishment of optimal breeding programmes requires the
appropriate use of genetic variation among and within breeds and the judicious use of hormone
products, which is an appropriate strategy in developing countries and more developed market
economies (Santos et al., 2015). This review is concerned with the use of hormone products in rams
to improve reproductive efficiency, which has implications for the productive economics of flocks
(Hashem and González-Bulnes, 2021). Scientific articles from the last 50 years, evaluating the use of
gonadotropin-releasing hormone (GnRH) in rams, were considered (Arroyo et al., 2016; Cardona-
Tobar et al., 2020). This approach differs from the use of "green" techniques and methods that have
been promoted to date, such as the use of the male effect and nutritional management in flocks.

Spermatogenesis in the ram

As a preamble to this review, a general description of spermatogenesis in rams is given,
followed by a focus on the hormones that have been used in recent years to increase ram
reproductive efficiency. Spermatogenesis, the process that transforms germ cells into
spermatozoa, is governed by mitotic and meiotic processes, going from cells with 54
chromosomes to 27 (Moraes et al., 1980; Cardoso and Queiroz, 1988). These mechanisms take
place in the seminiferous tubules, which protect and support the germ cells and in which the
spermatozoa are formed in their entirety, in a process that takes approximately 48 days in rams
as five cycles of approximately 10 days each are required (Courot and Ortavant, 1981). The
process is divided into three phases, which are regulated by the pituitary hormone system
(Figure 1). The hypothalamus secretes pulsatile GnRH, which stimulates the anterior pituitary to
produce and release gonadotropins: luteinising hormone (LH) and follicle-stimulating hormone
(FSH), the main players in the mechanisms of gametogenesis (Cardoso and Queiroz, 1988). As
hormonal mechanisms, they are controlled by various factors such as diet, light-dark hours, age,
heat stress and thermoregulatory processes (Cardona-Tobar et al., 2020). When released, LH
binds to Leydig cell receptors and stimulates the synthesis and secretion of testosterone (T) in
the inter-testicular space, binding Sertoli cells (SC) at the nuclear level. As FSH is also released, it
will bind to SC membrane receptors and work in conjunction with T to induce Sertoli cells to
produce androgen-binding protein (ABP), seminiferous fluid, androgen-binding proteins and
hormones. When FSH binds to Sertoli cells, it also stimulates the production of inhibin and activin,
exerting negative feedback with the pituitary axis and controlling the secretion of FSH and LH.
The FSH is required to initiate spermatogenesis at puberty, but once initiated, it is required to
initiate spermatogenesis only when inhibited by external factors (Wang et al., 2022; Ramaswamy
and Weinbauer, 2014). At puberty, which occurs in sheep occurs at around 4 months of age,
spermatogenesis begins because at this stage, the Leydig cells have already differentiated into
adult cells and contain receptors for LH and synthesise and release T, initiating spermatogenesis.

Figure 1. Hormonal regulation of spermatogenesis in rams.

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The signalling cascade and the mechanisms that carry out spermatogenesis are affected by
a number of factors that severely compromise the ram's reproductive performance. These
factors include heat stress, which causes fatigue, and dehydration, which adversely affects ram
development and production by activating thermoregulatory mechanisms such as thermal
shock, with negative impacts on food consumption and energy requirement by compromising
nutrition (Macías-Cruz et al., 2018b, c). Similarly, cell division-induced oxidative stress, which
consumes mitochondrial oxygen and makes the ram's reproductive system vulnerable, is also
produced by thermal, nutritional and seasonal stress, among others (Asadi et al., 2017).
Spermatogenesis begins at puberty, and its onset varies among in months among breeds, with
early age being the most favourable for sexual performance. However, although the number
of sperm decreases over the years, sperm cells do not disappear completely; notwithstanding,
the length of the seminiferous tubules decreases with age, affecting sperm production. In
parallel to the above, seasonality also plays a role since when the light-dark hours are affected,
the neuroendocrine that causes seasonal anoestrus is affected in most rams (Stewart et al.,
2021). In the following section, we look at the hormonal strategies that can be integrated to
reduce negative effects on spermatogenesis and improve semen quality in rams.

Sexual behaviour of rams

Through vast amounts of information presented by different studies involving the
analysis of male reproductive hormones, it has been confirmed that the sexual activity of
rams is under the control of androgenic hormones that act at the level of the hypothalamus
(preoptic area) (see complete reviews of Orihuela-Trujillo, 2012). One of the main hormones
released by rams is testosterone, which influences sexual and metabolic activity and varies
throughout the year; it is present at low concentrations outside the reproductive season,
while testosterone levels increase above the threshold required for the ram to show
sufficient sexual behaviour to detect a female in oestrus (Bahadi et al., 2023) and induce her
to cover and become pregnant within the same season. Ungerfeld (2021a) systematically
characterises sexual behaviour and describes the main scenarios that can influence sexual
development from lambing to adulthood. The success of any sheep breeding programme
depends on three important reproductive aspects of the ram: libido (interest in mounting a
female), mating ability (ability to cover ewes in heat) and semen quality (fertility) (Price, 1987).
These three key points are required to ensure that each of the rams involved in a mating
programme will have interest in covering an oestrus ewe and will present good semen
quality. Similarly, rams with poor sexual libido which show poor competition for an oestrous
female will produce fewer offspring in future generations. In this case, the component known
as sexual behaviour in rams has been the subject of much research under different scenarios
to determine whether it contributes significantly to the effectiveness of the male effect
(Perkins and Fitzgerald, 1994). The same authors compared high and low sexual behaviour
rams when exposed to Targhee and Ramboulliet wool ewes; they found that a high
percentage of ewes ovulated when exposed to high sexual behaviour rams (95%) compared
to ewes exposed to low sexual behaviour rams (78%).
The aim of this review is not to describe all of the factors involved in or influencing sexual
behaviour and the male effect, as comprehensive reviews have been previously undertaken
over the years (see full reviews by Pretorius, 1972; Tilbrook and Cameron, 1990; Rosa and
Bryant, 2002; Ungerfeld et al., 2004; Perkins and Roselli, 2007; Orihuela-Trujillo, 2012; Roselli,
2020; Ungerfeld, 2021a, b). However, it is necessary to mention that consistent studies have
been carried out over the years investigating factors that influence sexual behaviour (see
Figure 2), where the different factors that can influence both wool and hair rams have been
shown (Bahadi et al., 2023). It is important to bear in mind that there are differences which, in
one way or another, affect the mating of a ram to an oestrus ewe to a greater or lesser extent,
as well as the achievement of optimal gestation of two offspring. Based on these factors,
studies have been performed to evaluate different hormones used to improve the sexual
behaviour of rams in the different scenarios highlighted in Figure 2.

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Figure 2. Factors influencing sexual behaviour in rams (as mentioned in the reviews by Orihuela-Trujillo,
2012; Ungerfeld, 2021a, b).

Reproductive seasonality of rams

Among these factors, the one that most influences sexual behaviour is related to
seasonality, as most breeds are seasonal and show a higher libido when natural light decreases
(short days). This reproductive symphony is controlled by the photoperiod (see Figure 3). In the
cited literature, it is confirmed that rams are less affected than ewes, but maintain their fertility
(Santos et al., 2015; Zaher et al., 2020; Hedia et al., 2020; Kleeman et al., 2021), especially in
hair rams (Sánchez-Dávila et al., 2020; de Sá Geraldo et al., 2024). However, different hormonal
strategies have been evaluated to improve the sexual activity of rams in and out of the
breeding season, which are described in the following sections.

Figure 3. RHT: Retinohypothalamic tract, SCN: Suprachiasmatic nucleus, PVN: paraventricular nucleus,
SCG: superior cervical ganglion, PG: Pineal Gland, NA: Noradrenaline, DA: Dopamine, SHT: Serotonin,
GnRH: Gonadotropin-releasing Hormone, LH: Luteinising hormone, FSH: Follicle-stimulating hormone.

On the other hand, with regard to the physiological differences that may exist between hair
and wool rams from a reproductive point of view, it should be mentioned that the reproductive
seasonality is longer and more pronounced in wool rams, and they show a longer seasonality
due to their greater body weight, with each breed responding to the photoperiod depending
on the latitude at which they are commercially exploited (Ungerfeld, 2012). In this case, woolly
males show seasonal variations in semen quality and sexual activity, the duration of which is
less pronounced compared to ewes (Mamontova et al., 2021; Aibazov et al., 2022).
Consequently, woolly sheep breeds are characterised by different photoreactivity, where the
effect of seasonality and circadian variations on sexual behaviour and semen quality in rams
has been studied from tropical latitudes to both poles of the earth (Godfrey et al., 1998;
Mamontova et al., 2021). On the other hand, with regard to hair rams, recent studies have been
carried out on the influence of seasonality on their reproductive physiology, where it was found
that they can show variations, such as in semen quality, but not reach zero values, for example

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in Pelibuey and BlackBelly rams (Aguirre et al, 2007; Cárdenas-Gallegos et al., 2012), Saint Croix
rams (Sánchez-Davila et al., 2020) and Santa Inés rams (de Sá Geraldo et al., 2024); all studies
concluded that the seasonal dimension was not sufficient to prevent rams from being able to
cover and gestate ewes at different times of the year. Similarly, in terms of sexual behaviour,
large differences can be found between wool and hair breeds, where wool rams can reach no
or low sexual activity (Avdi et al., 2004; Mamontova et al., 2021) compared to hair rams, which
are mainly used at latitudes below 22° and show significant variations in sexual behaviour
when there are high ambient temperatures and relative humidity (Aké-Villanueva et al., 2019;
Sánchez-Davila et al., 2020; de Sá Geraldo et al., 2024). For example, the use of hair rams to
stimulate Suffolk wool ewes during the anoestrus period achieved up to 91.66% of ewes being
covered and lambed when mounted by Saint Croix rams (Clemente et al., 2012). Therefore, the
use of hormonal strategies is currently focused on eliminating the effects of seasonality, mainly
in wool rams; in contrast, the aim in hair rams is to minimise the effect of environmental factors
in order to increase the reproductive efficiency of males in any mating season.

Administration of hormonal products to stimulate sexual activity and

spermatogenesis in rams
At present, the use of hormonal strategies to improve semen quality and sexual behaviour
in rams is mainly based on six of the most important hormones that have been studied to
improve the reproductive performance of rams and have a positive impact on flock
productivity. The scientific literature was searched for the most representative publications for
each of the hormones described in this review. This is the first review that collectively describes
the hormones that have influenced the scientific development of ram reproduction over 50
years or more. There is currently a great deal of research, including reviews, on each of these
hormones, but they have not previously been described together. In addition to the above
productive advantages, hair rams do not exhibit the pronounced seasonal reproduction seen
in wool breeds (Macías-Cruz et al., 2009). Although semen quality and reproductive activity are
reduced, the ability to mate and produce semen is maintained throughout the year
(Stewart et al., 2021). Currently, various hormonal alternatives are being sought to increase
sexual activity and semen quality in rams as this is a novel research field and has not been fully
explored. Over the past decade, hormones such as cloprostenol (Daham, 2012) and T
propionate (Calderón-Leyva et al., 2019), prostaglandin analogues and equine chorionic
gonadotropin (eCG), as well as kisspeptin (Daniel et al., 2015), melatonin (Pool et al., 2020b)
and oxytocin (El-Shalofy and Hedia, 2021b), have been investigated in rams.

Kisspeptin (Kp)

Successful reproduction control, at least in ruminants and especially in sheep and rams,
requires the integration of external and internal factors so that the pulsatile secretion of
GnRH by the hypothalamus can take place; the most important factors are the feedback of
sexual steroids, feeding and body condition, breeding season, pheromone production, stress
and age of the animals (Scott et al., 2019; Harman and Serpek, 2022). Kisspeptin, also known
as metastin, inhibits the metastasis of cancer cells (Daniel et al., 2015). In 1971, it was
discovered that the hypothalamic-pituitary-gonadal axis is responsible for regulating
reproduction in mammals, but the positive and negative feedback mechanisms that regulate
the pulsatile secretion of GnRH were not elucidated until 2003 with the discovery of the set
of neurons called kisspeptins, one of the most important discoveries in relation to
mammalian reproduction (Asadi et al., 2017; Abou-Khalil and Mahmoud, 2020;
Meccariello et al., 2020). Kisspeptin and its receptors also play an important role in the
regulation of the reproductive axis; kisspeptin neurons are produced in the preoptic area
(POA) and arcuate nucleus (ARC) neurons and act directly on GnRH neurons, stimulating
gonadotrope cells to release LH and FSH and acting directly on the male gonads of mammals
(Meccariello et al., 2020). Kisspeptin is an important neuropeptide involved in the onset of
puberty, the regulation of fertility, the secretion of gonadotropins and the proper functioning

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of the gonads (Beltramo and Decourt, 2018; El-Sherry et al., 2020). In addition, it is a regulator
of the male reproductive system with a wide range of functions; improves semen quality,
including sperm viability and concentration, and increases testicular T levels (Abou-Khalil and
Mahmoud, 2020; Beltramo and Decourt, 2018). Kisspeptin has been detected in the
epididymal epithelium, and its receptor is expressed in the acrosomal region of both
spermatids and mature sperm (Abou-Khalil and Mahmoud, 2020). This renders the sperm
capable of fertilisation during transfer through the epididymis and into the female
reproductive tract (Meccariello et al., 2020). Kisspeptin has also been found in Leydig and
Sertoli cells (Beltramo and Decourt, 2018). Spermatids and mature spermatozoa possess a
receptor for kisspeptin and are exposed to kisspeptin-synthesising cells from the
seminiferous tubules to the female reproductive tract. Thus, kisspeptin is part of the
microenvironment of spermatozoa and mediates their maturation (Abou Khalil and
Mahmoud, 2020).
However, especially in zootechnically important animals, endogenous Kp has limitations
in terms of its short half-life and poor pharmacodynamics, limiting its use in large-scale
experiments (Seminara, 2006). Recently, synthetic KISS1R agonists based on the kisspeptin
scaffold have been developed, which enabled studies on their use. For example, EL-Sherry
et al (2020) which showed that the intramuscular application of 5 µg/kg live weight of Kp in
Ossimir rams increased the ejaculate volume, sperm concentration and percentage of live
sperm. Similarly, the trajectory and rheotaxis of spermatozoa were improved by the use of
Kp, which appears to influence the neuroendocrine control of reproduction, at least in
ruminants, culminating in the secretion and metabolism of LH (Daniel et al., 2015). Kisspeptin
is encoded by the KISS1 gene and acts through GPR54 (Kiss1r) to induce GnRH secretion
(Seminara, 2006). Because of its importance in the brain, Kiss1r is expressed by GnRH
neurons. Administration of Kp to ruminants stimulates the release of LH and FSH (see
Figure 4) (Harman and Serpek, 2022; Abou-Khalil and Mahmoud, 2020; Abdel-Ghani and
Mahmoud, 2019). The intra-testicular effect of Kp on T production and spermatogenesis in
ruminants is the subject of ongoing research.

Figure 4. Schematic representation of the mechanisms controlling reproduction in rams.

Abou Khalil and Mahmoud, 2020 mention that treating rams aged 1.5–2 years with a dose
of 5 mg/kg body weight of kisspeptin-10 (KP-10)/week for 1 month improves testicular weight,
which depends on sperm development and may be highly related to T concentration. This leads
to an improvement in the histological characteristics of the testes caused by the proliferation
and differentiation of KP-10. In the same experiment, treatment with KP-10 had a direct effect
on the significant improvement in semen quality, including scrotal circumference. In studies in
prepubertal cattle, Kp stimulated the secretion of LH and FSH (Ezzat et al., 2010), and in
monkeys, it stimulated the secretion of LH, FSH and T (Ramaswamy and Weinbauer, 2014). In
a more recent study carried out in bucks, where kisspeptin-10 was evaluated at doses of 4 and
8 µg/kg/animal outside the reproductive season, the T concentration increased 50 minutes

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after the application of both doses (Al-Ameri et al., 2019). In a study by Rietema et al. (2019),
more protein cells were found in the mid-dorsomedial hypothalamus and mid-arch when the
rams were sacrificed, suggesting that they mediate the increase in GnRH and LH production
due to acute nutritional supplementation, which may be more pronounced when rams are
given acute protein supplementation (30 g kg-1 bw per day) as it activates Kp neurons in the
arcuate nucleus of the brain.

Oxytocin
Oxytocin (Ox) was first described as a uterotonic agent in 1906 and first synthesised in
1953 (Stadler et al., 2020). It is a hormone that is routinely used on dairy farms
(Rietema et al., 2019). Oxytocin is stored and released by the posterior pituitary of the
hypothalamus and is a peptide neurohormone; its receptors are found in the brain, pituitary
gland, heart and gonads (Rietema et al., 2019; Stadler et al., 2020; Knickerbocker et al., 1988).
This hormone contracts the smooth muscle of the uterus and mammary gland, which is
important for sperm transport after copulation as well as facilitating labour and udder
contractions (Stadler et al., 2020). Similarly, Ox is produced by Leydig cells, the epididymis
and the prostate, where its receptors are found (Knickerbocker et al., 1988), and increases
the contractions of the seminiferous tubules and the walls of the epididymal ducts, thus
facilitating the transfer of spermatozoa (Stadler et al., 2020; Assinder et al., 2000;
Dalmazzo et al., 2019; Ruan et al., 2020). It is frequently used today, especially in male
reproduction in small ruminants, in different aspects, such as for semen collection with the
electro-ejaculator, where, when used together with the prostaglandin (PG) before semen
collection, it reduces the time and number of electrical stimuli required to obtain a semen
sample without affecting its quality, both in goat bucks (Ruan et al., 2020) and rams (Abril-
Sánchez et al., 2019; Luna-Palomera et al., 2021).
Considering that Ox increases sperm transport by increasing epididymal contractility and
that is an effective and economical adjunct to improve at least one important characteristic of
semen quality, (Stadler et al., 2020; Luna-Palomera et al., 2021), a dose of 10 IU of this hormone
can increase the ejaculate volume in semen collection in rams using an artificial vagina.
However, it is important to note that equally important characteristics such as sperm
concentration, motility and serum T concentration were not affected, which relegates this
treatment to an option to increase semen volume only.
Oxytocin is produced locally in Leydig cells and is under the control of LH and lipoproteins
but not T (Stadler et al., 2020). It regulates basal epididymal contractility and stimulates
sperm release from the storage site in the epididymis at the time of ejaculation
(Knickerbocker et al., 1988; Dalmazzo et al., 2019; Stadler et al., 2020). Application of Ox prior
to semen collection can increase sperm concentration in several mammalian species
including rams, rabbits, cattle and buffalo (Luna-Palomera et al., 2021). Therefore, it seems
reasonable that Ox is involved in the transition of sperm through the male ducts and may
accelerate ejaculation (Knickerbocker et al., 1988; Abril-Sánchez et al., 2019). In this sense,
Ox can help improve semen quality. In rats and sheep, exogenous Ox administration affects
the contraction of the seminiferous tubules to promote the exit of sperm towards the rete
testis (Da et al., 2013; Stadler et al., 2020). Another study showed that Ox can regulate basal
epididymal contractility while stimulating sperm release during ejaculation (Luna-
Palomera et al., 2021).
In rams, exogenous Ox application can increase the concentration of spermatozoa
(Assinder et al., 2000). This has been verified by immunocytochemical studies: when a dose
of 20 µg of Ox was applied to adult rams, Ox receptors could be identified in Leydig and
Sertoli cells as well as in the epithelial cells of the epididymis, thus confirming that Ox can
modulate sperm transport, which is supported by the location of Ox receptors in the tail
of the epididymis and the vas deferens, thus regulating the steroidogenesis of the ram (Jung
and Yoon, 2021). However, at least in male goats, administration of 0.7 IU/kg Ox increased
testicular blood flow (vasodilator effect) and decreased plasma T concentrations (control
of stereodigenesis) 60 minutes after application, which can be explained by the inhibition

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of the formation of the C21 and C19 metabolites of pregnenolone and, therefore, T
concentrations (Hedia and El-Shalofy, 2022). These differences may be partly due to the
different species and doses studied. Regarding seminal quality in rams, the administration
of low doses of Ox (5 IU) increased the volume, motility and sperm concentration at 10
minutes after application (Narenji Sani et al., 2014). In males of other domestic species,
such as rabbits (Fjellstrom et al., 1968), cattle [(Berndtson and Igboeli, 1988) and buffaloes
(Ibrahim, 1988), there is also evidence of a positive effect of Ox on semen quality
(Tiptanavattana et al., 2022). In rams, however, it must be considered that the contractile
effect of Ox would lead to the emptying of the spermatozoa stored in the epididymis,
resulting in an increase in the number of abnormal spermatozoa after the 6th week, which
is the period required for spermatogenesis (Stadler et al., 2020). A study in Mexico
evaluated the effect of the administration of exogenous Ox prior to semen collection with
an artificial vagina in Pelibuey rams (Luna-Palomera et al., 2021); the application of 10 IU
intramuscularly significantly increased the semen volume by 30% compared to that of rams
not treated with Ox, but without a significant effect on semen quality variables such as
total sperm concentration and sperm per mL. Another aspect evaluated in this work was
the effect of 10 or 20 IU of Ox 20 minutes after administration on T levels, with no
significant differences found. The results described above contrast with those of previous
studies (Nozdrachev et al., 1994; Inaba et al., 1999) that investigated the relationship
between exogenous Ox and other hormones. However, the effects of Ox on T are still
subject of debate as some authors reported that Ox treatment reduces the T concentration
(Berndtson and Igboeli, 1988) or has no impact on it (Ibrahim, 1988), whereas others
(Nozdrachev et al., 1994) report that it increases T release. In terms of sexual behaviour,
the application of 10–20 IU of Ox in goats has a positive effect on libido and reduces the
time needed to collect semen samples (Çiğdem et al., 2019). This is due to the interaction
of components involved in male sexual behaviour, such as dopamine, serotonin, amino
acids and Ox, which act directly on the paraventricular nucleus of the hypothalamus (Harris
and Nicholson, 1998; Argiolas and Melis, 2004; Bozkurt et al., 2007). In a descriptive way,
Table 1 presents the most recent summarised studies on the use of Ox in rams.
It shows the main values obtained from each of the studies cited; 62.5% of these studies
were carried out during the reproductive period, mostly in hair breeds. Regardless of the
application route and the doses used prior to semen collection, it is standardised that Ox has
a main effect on ejaculate volume and increases the T concentrations (Anjum et al., 2018). This
is especially true in the first 10 min after application, regardless of the route of administration
(IM or IV). Another interesting point is that there are positive effects on ejaculate volume and T
levels, regardless of the method of semen collection (Stadler et al., 2020).
In recent years, Ox has been used in combination with PGF2α to obtain semen samples by
the electroejaculation technique and to reduce the stress linked to this technique
(Palmer et al., 2004; de Moura Fernandes et al., 2021). In this sense, Ox modulates the
contractility of the ram's reproductive tract and affects sperm transport and maturation as well
as spermiation (Assad et al., 2016). For example, in a study conducted in male goats, the
combined use of a PGF2α analogue (cloprostenol, 250 μg IM) and Ox (10 IU IM) 5 minutes and
30 seconds before electrical stimulation respectively, decreased the number of pulses and
tended to decrease the number of vocalisations presented by the goat bucks receiving this
treatment; seminal quality was not affected (Ungerfeld et al., 2018). The same effect was
reported in rams treated with the same doses of PGF2α and Ox analogue, where the
administration of GnRH (4.2 μg of buserelin acetate) increased the T levels (de Moura
Fernandes et al., 2021). Similarly, another recent study using an Ox analogue (carbetocin, 0.1
mg) via IV found limited effects on the time required for semen sample collection in bucks and
only an effect on respiratory frequency in Iberian ibex (Ungerfeld et al., 2023). These new
studies on the use of Ox alone or in combination with PGF2α require further studies on the
frequency and dose, also outside the reproductive season, which means that the males might
show high semen quality but without sexual behaviour.

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Table 1. Summary of the studies on oxytocin, including breeds, doses, method of administration, time of
the study and the main effects.

Collection time
Breeding Application Collection
Reference Breed after Dose Main effects
season method method
application
Testosterone
decreased 60
0.7 IU/kg minutes post-
at 0, 5, 30, treatment.
El-Shalofy and 12 Baladi
Within IV Does not apply Does not apply 60 and Oestradiol 17-β
Hedia (2021a) goat bucks
120 not affected.
minutes Increased
testicular blood
flow
Testosterone and
oestradiol-17β
increased from 5
to 30 minutes after
Single
El-Shalofy and 16 Ossimi injection and
Within IV Does not apply Does not apply dose of
Hedia (2021b) rams decreased until
20 IU
120 minuates.
Increased
testicular vascular
tone
Increase in the
number of
spermatozoa and
seminal fluid.
Cannulation
Nicholson et al. Not 24 Oxford 10 and Promoted an
IV of the Each 10 min
(1999) reported Down rams 100 μg increase in the
epididymidis
transport of
spermatozoa in
the vas deferens
and the ejaculate
After 24 hours, Increased the
Castration of the Single
Whittington et al. 15 Oxford semen samples concentration of
Within IM rams after dose of
(2001) Down rams were extracted spermatozoa in
application 20 μg
from the testicles the rete testis
Collecting the
semen sample
20 minutes after
the administration
of 20 IU oxytocin
Çiğdem et al. 20 Norduz 10 and did not improve
Within Artificial vagina
(2019) bucks 20 IU overall semen
quality. However,
libido, semen
volume and sperm
concentrations
were increased

Gonadotropin-releasing hormone (GnRH) agonists

The GnRH is a hormone produced by neurons in the arcuate nucleus of the hypothalamus. It
has an endocrine function and is transported via the portal system to the pituitary gland, activates
its own receptors and stimulates the release of FSH and LH via the pituitary gland so they can
perform their functions in the ovaries and testes (see Figure 3) (Adams, 2005; Zhao et al., 2023). In
general terms, GnRH is a decapeptide comprising 10 amino acids arranged sequentially. It is a
neurohormone as it is produced by neurons. The gene encoding the GnRH precursor is situated on
Chromosome 8 in mammals (Foster et al., 2006; Zhao et al., 2023), and GnRH is secreted in pulsatile
form and released into the portal system in pulses of different amplitudes and frequencies. Its half-
life is short (2–4 minutes) (Zhao et al., 2023). In both goats and sheep, high GnRH activity levels
commence during puberty and continue throughout the reproductive lifespan (Foster et al., 2006).

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As a result of identifying the activity of GnRH, comparable molecules, known as analogues, have
been developed synthetically. To imitate the effect of GnRH and to bond tightly with its receptors
in the pituitary gland, these molecules replace some amino acids within the initial GnRH molecule
(Moenter and Evans, 2022). The GnRH analogues are capable of obstructing the natural GnRH
activity and hindering the release of FSH and LH from the pituitary gland to the gonads (Adams,
2005). This function has been leveraged to create medications for use in assisted reproduction
therapies, artificial insemination and in vitro fertilisation (Hawken and Martin, 2012).
Two types of GnRH analogues have been developed, with different mechanisms of action.
The first type are GnRH agonists, which have a longer half-life and a high affinity for the GnRH
receptor. Binding of the agonist to the receptor results in an abrupt release of gonadotropins,
known as the "flare-up" effect, which is a sudden increase in LH release and used in
reproductive programmes to synchronise ovulation (Moenter and Evans, 2022). However, a
drawback of these GnRH agonists is that with continuous administration, the receptors
become obstructed and unresponsive, resulting in the suspension of FSH and LH secretion
(Adams, 2005). However, GnRH antagonists are more directly inhibitory in their effect as they
block the receptor immediately upon binding (Amodei et al., 2022; Hitesh and Kansal, 2022;
Hashem et al., 2023). Moreover, in goat and sheep production systems, farm productivity must
be based on the continuity of kidding and lambing throughout the different seasons.
Therefore, since the 1980s, the priority for both species has been to increase the number of
continuous births and to improve lamb and kid yield as well as milk production. To this end,
natural strategies such as light programmes for males and the use of melatonin implants have
been developed, based on the knowledge of the male effect in both species, to significantly
improve economic indices outside the non-breeding season. Although these techniques are
effective, the males take time to respond. In this case, as with the previous hormones, GnRH
agonists have been used in zootechnically important males to release T (Do Espìrito
Santo et al., 2022, Yousef et al., 2022). In females, their use has been extended to oestrus and
ovulation synchronisation programmes as well as multiple ovulation and embryo transfer
(MOET) programmes. However, they are now starting to be used in small ruminant males as
there is not sufficient information on females (Prestel et al., 2022).
In the late 1960s and 1970s, the application of GnRH to enhance semen quality and stimulate
T release in rams was first explored. Schanbacher and Lunstra (1977) evaluated the use of 50 µg
twice daily for 7 weeks and provided evidence of an advantageous effect on sexual behaviour, as
measured by a mating index score, with 60% of the rams treated with GnRH successfully
completing the 20-minute behavioural test. In a recent study conducted in bucks, the
intramuscular administration of a GnRH analogue (buserelin acetate = 4.2 µg/animal/10 days)
resulted in a significant increase in T concentrations, particularly in the initial hours after
administration. Improvements were also observed in certain semen quality variables such as
mass motility as well as the percentages of motile and normal sperm. It was concluded that,
outside of the reproductive season, the short-term administration of buserelin increases T levels
and enhances sperm quality without impacting the secretion of gonadotropins from the
pituitary-gonadal axis and, consequently, the T levels (Giriboni et al., 2019). Another study
indicated that, at least in dogs, using GnRH could rectify sperm dysfunction through
subcutaneous administration of three injections. These should be administered once per week
at a dose of 1 µg/kg of buserelin acetate. However, it should be noted that the extended usage
of GnRH inhibits LH and T secretion by the anterior pituitary and testes (Kawakami et al., 2005).
A strong effect of GnRH occurs shortly, after the first hours of application.
In a study conducted on Ossimi rams, applying a single dose of GnRH (buserelin acetate:
0.008 mg/ram) intravenously resulted in an increase in T levels during the first 3 hours and a
progressive increase in oestradiol-17β levels from the 3rd to the 120th hour of application (El-
Shalofy and Hedia, 2021a). However, it should be noted that the excessive use of the agonist
buserelin acetate may result in negative feedback effects. In Santa Inés rams, the T levels
peaked at week after administering 2.5 µg of buserelin acetate (Do Espírito Santo et al., 2022).
Additionally, in bulls, applying 100 µg/48 h can preserve the scrotal thermoregulatory system's
function without impacting semen quality (Romanello et al., 2018). These studies have
demonstrated that although the LH secretory responses decreased after single or repeated

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injections of GnRH, maturation of the testes' steroidogenic capacity occurred. The increased
steroidogenic activity may have been a direct result of the increased number of Leydig cell
receptors (Wilson and Lapwood, 1979).
To summarise the action of GnRH in the regulation of reproduction, the animal's own
intrinsic and environmental factors influence the hypothalamic-pituitary-gonadal axis,
resulting in the pulsatile secretion of GnRH from the hypothalamus. Therefore, in view of the
global legislative requirements to provide better animal welfare, it should be taken into
account that the increasing use of more natural diets and such that affect the nutritional status
of animals and limiting the use of steroid hormones in reproductive processes and the
secretion of FSH and LH to regulate endocrine function and increase ram reproductive
efficiency are important measures. It is therefore certain that the prolonged use of GnRH
agonists will significantly reduce serum T concentrations, testicular volume and
spermatogenesis Adams (2005), which confirms the current importance of research aimed at
determining the optimal dose of GnRH agonists in rams under different environmental
scenarios, with emphasis on nutrition, age and breed, among other factors.

Equine chorionic gonadotropin (eCG)

eCG is a high molecular weight glycoprotein derived from the endometrial cups of the
mare's uterus (produced from 35-100 days of gestation); it is extracted from the blood of
pregnant mares. In these females, eCG has LH activity (Luna-Palomera et al., 2019), but in the
cow it has activity as FSH or LH, depending on the receptor populations in the ovarian follicles
at the time (De Rensis and López-Gatius, 2014; Murphy, 2018; Bo and Menchaca, 2023).
Regarding eCG in rams, recent studies conducted by a group of researchers from South
America (Uruguay) evaluated the application and evaluation of eCG in small ruminants (bucks
and rams) under different scenarios. Their research was published in high-impact journals,
giving validity to the results obtained. Table 2 provides a summary of the work carried out
mainly by this group. According to the results, the use of eCG within and outside the breeding
season affects T concentrations and sexual behaviour but not semen quality and scrotal
circumference. In rams and bucks, it has been used since 2008, when doses of 100 to 400 IU of
eCG were evaluated in prepubertal lambs (Ungerfeld and Bielli, 2008), with no effect on semen
quality and sexual behaviour; However, at does higher than 400 IU, up to 1000 IU, or when two
doses of 1000 IU are given to rams 3 days before mating with ewes, it can induce an increase
in T secretion in rams during the period of sexual rest (Ungerfeld et al., 2018; Ungerfeld et al.,
2019), but without altering semen quality (Beracochea et al., 2020). In this sense, eCG has been
used to increase libido and make the use of the male effect more efficient in rams
(Ungerfeld et al., 2014, 2019). Similarly, its use has been extended to bucks, as in rams, but with
an initial dose of 800 IU of eCG and four doses of 500 IU applied every 5 days during the period
of sexual rest, resulting in an increase in T secretion and an improvement in the quality of fresh
semen, with an increase in the level of eCG antibodies (Beracochea et al., 2018).
Based on the literature, eCG has an effect on the increase in T concentration, and only two
articles mention that it has an effect on seminal quality. Specifically, Narenji Sani et al. (2014),
using Zel rams and eCG doses of 400 and 600 IU, and Beracochea et al. (2020), reported
increased cryopreservation rates (see Table 2). Notably, it is difficult to obtain high-quality
semen from rams treated with eCG as it has a half-life in the blood of 63 hours due to its high
content of sialic acid, a sugar that is incorporated into eCG proteins during glycosylation in cells
and acts as an intercellular signalling agent (Aguirre-Zapata et al., 2023). Recent studies have
used the combined application of eCG (400 IU) with other hormones, such as melatonin (18-
mg melatonin implant), for 18 weeks in male goats. Based on their results, there was a decrease
in non-viable and morphologically abnormal spermatozoa and an increase in T concentrations
and semen quality, observed from the 3rd week of the study (see Table 3). (Abbas et al., 2021).
These results are interesting because of the combination of the two hormones: on the one
hand, eCG, which can directly increase the level of intra-testicular T, directing its action to the
differentiated spermatozoa found in the epididymis and in the accessory sex glands, where the
eCG receptors are located (Min et al., 2004), causing a change in the composition of the seminal

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plasma (Matsuoka et al., 2006); on the other hand, melatonin, which has the limitation that it
can exert its effect only up to 35 days after its application (Abbas et al., 2021). Therefore, its use
may be limited in terms of the frequency of application to have a positive and lasting effect on
fertility in rams; however, it may be limited by the production of antibodies, although this does
not appear to be the case, at least in male goats (see Table 2) (Beracochea et al., 2018).

Prostaglandins
The use of PGs started in the last century (1930), when it was observed that the uterus
contracted in response to human seminal plasma (Capitan et al., 1990). This led to a wave of
research and extensive literature, with the recent discovery of PGF2α. Today, prostaglandin
F2α analogues are hormones used extensively in farm animals to control and synchronise the
oestrous cycle in females, particularly in the luteal phase, due to their effects on the ovary,
causing luteolysis (Fierro et al., 2013). Prostaglandins are bioactive molecules derived from
arachidonic acid. Their biosynthesis is initiated by the action of cyclooxygenase (COX)
(Frungieri et al., 2018), which converts arachidonic acid to PGG2, which is further reduced to
PGH2 by a peroxidation reaction also catalysed by COX (Echeverrìa, 2006). Two commercial
analogues are commonly used experimentally in domestic animals. One is cloprostenol, which
was synthesised in 1970 as part of a project to develop PG analogues with potent luteolytic
activity (Ramìrez et al., 2018). The other is dinoprost tromethamine, developed in the late 70s
of the 20th century. The main characteristic of this substance is that it is similar to that produced
naturally in the uterine tract (Moreira and Hammon, 2012; Ramìrez et al., 2018). It is a white
salt, crystalline in appearance and hygroscopic in nature, with effects similar to those of PGF2α,
in particular luteolytic and vasoconstrictive effects, and is rapidly metabolised by the
respiratory system after a few minutes (Sakamoto et al., 1995).
The mechanism of action of prostaglandins is intrinsically linked to specific receptors; these
receptors activate a specific G-protein which triggers the AMPc cascade and the corresponding
release of calcium via phosphatidylinositol (Echeverrìa, 2006). They are located on the cell
membrane and classified according to the type of prostaglandin they bind.
To date, nine receptor types have been identified, namely DP1-2, EP1-4, FP, IP and TP,
referring to the receptor that binds the corresponding prostaglandin, with FPs binding to
PGF2α (Grösch et al., 2017). Several authors have reported a beneficial effect on ejaculate
volume and sperm concentration in dogs (Traas and Root-Kustritz, 2004), bulls (Titiroongruang
and Tummaruk, 2011) and bucks (Daham, 2012), which can be explained by the movement of
sperm from the epididymis into the vas deferens as a result of the aforementioned
vasoconstrictive effects.
The mechanisms of action of prostaglandins on male sexual behaviour are not fully
understood, but to date, various authors have proposed hypotheses in other species, such as
pigs, indicating that PGF2α stimulates some parts of the central nervous system or at least
interacts with the hypothalamus, which is one of the regulators of the central nervous system
(Estienne, 2014). In cattle, they have been associated with a positive feedback effect on the
hypothalamic-pituitary-testicular axis (Sakamoto et al., 1995), and in rodents, they are involved in
germ cell development and steroidogenesis (Titiroongruang and Tummaruk, 2011). At the
physiological level, in studies on yearling bulls, serum T levels doubled when PGF2α was
administered and remained at high levels for a period of 4 hours (Titiroongruang and Tummaruk,
2011). It has been suggested that PGF2α directly stimulates testicular steroidogenesis through
the production of cyclic AMP (Estienne, 2014; Grösch et al., 2017). To date, and in the absence of
more detailed studies on the effects of PGF2α, it is believed that it directly acts on the contractile
tissues from the testicular capsule and epididymis to the vas deferens.
The use of PGF2α analogues prior to collection can optimise the number of spermatozoa,
as shown in Table 3, where two analogues have been used in recent years, namely cloprostenol
dextrogy and dinoprost tromethamine. Regardless of the breed, species and doses used, there
is a positive effect on ejaculate volume, mass motility and sperm concentration in goats, bulls
and rams. However, due to the consistent lack of studies on rams, few studies have
investigated the use of these analogues and their effects on sexual behaviour. For example,

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regarding the use of PGF2α analogues in hair rams, Ungerfeld et al. (2020) reported that the
administration of 10 mg dinoprost tromethamine in adult Saint Croix rams had no positive
effects on the main variables of sexual behaviour, which limits their application. In another
study (Sánchez-Davila et al., 2022) conducted in Katahdin lambs with no previous sexual
experience, the use of PGF2α analogues had a positive effect on sexual behaviour by reducing
the time to initiate courtship and increasing the frequency of anogenital sniffs, flehming and
lateral approaches (Table 3). Specifically, animals treated with dinoprost had a higher number
of mounts, whereas those treated with cloprostenol had a higher mounting efficiency,
requiring fewer mounts to ejaculate. The same study reported that the effects of D-
cloprostenol on semen quality were highly relevant as the group treated with this drug
obtained more volume and greater mass motility per ejaculate than their counterparts treated
with dinoprost and the control group; there was also an upward trend in sperm concentration
in this group (Table 3) (Sánchez-Davila et al., 2022).

Table 2. Summary of the studies on eCG, including breeds, doses, method of administration, time of the
study and the main effects.

Collection
Breeding Application Collection
Reference Breed time after Dose Main effects
season method method
application

Increased semen
Narenji 18 Zel Single dose 0,
Outside IM Electroejaculator 10 minutes volume, mass activity,
Sani et al. (2014) rams (Iran) 400 IU, 600 IU
total number of sperm

Testosterone
First study: concentration
20 Saint Does not Two doses of increased in rams
Within IM Does not apply
Croix rams apply 1,000 IU of eCG given eCG both within
Ungerfeld et al. on days 0 and 4 and outside the
(2018) breeding season.
2nd study: Better sexual
10 Saint Does not Two doses of behaviour within the
Outside IM Does not apply
Croix rams apply 1,000 IU of eCG breeding season in
on days 0 and 4 rams given eCG.
Administration of eCG
affected testosterone
concentration,
antibody titres, the
Initial dose:
percentage of motile
800 IU (day 0),
spermatozoa and
Beracochea et al. 19 Gabon -14 to 56 followed by
Outside IM Electroejaculator spermatozoa with
(2018) bucks days four doses of
progressive motility
500 IU every
and the percentage of
5 days
spermatozoa with a
functional membrane
throughout the study
period
The anti-eCG titre was
higher in treated
bucks
(181.7 ± 61.3 ng/µL) vs
control bucks
Initial dose:
(31.1 ± 10.7 ng/µL).
800 IU (day 0),
No significant
Beracochea et al. 19 Gabon followed by
Outside IM Electroejaculator 91 days differences among
(2020) bucks four doses of
treatments in
500 IU every
testosterone
5 days
concentration, fresh
and thawed semen
characteristics, scrotal
circumference and
sperm cryoresistance

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Table 2. Continued…
Collection
Breeding Application Collection
Reference Breed time after Dose Main effects
season method method
application
Testosterone
concentrations were
First study: statistically higher in
Three doses rams treated with 700
-4, -1, 3, 6, 9, every 6 days: 0, IU eCG than in rams
27 Merilin
IM Electroejaculator 12, 15 and 400 and 700 IU. treated with 0 or 400
rams
22 days Day 0 = day of IU eCG. Testicular
the first characteristics and
administration. semen quality did not
differ among
treatments
Beracochea et al.
Outside The use of eCG had no
(2020)
effect on scrotal
circumference and
fresh semen
Second study:
characteristics. The
15 -7, -5 and two doses of
ratio of
Highlander; IM Electroejaculator weekly for 3 1,000 IU of
cryopreservation of
17 Texel weeks eCG: days 0
motile and progressive
and 5
spermatozoa was
higher in rams treated
with eCG compared to
the control group
Testosterone
concentration
T1 = melatonin
increased from
implant of 18
week 3 for T1
mg and 400 IU
(4.2 ± 0.2), followed by
of eCG
T2 (1.2 ± 0.1) and T3
(18 injections
(0.28 ± 0.1). Melatonin
every 4 days)
levels also increased at
week 5 in T1
Abbas et al. 9 Beetal Subcutaneous
Artificial vagina (12.5 ± 0.2), followed
(2021) bucks and IM
by T2 (10.2 ± 0.1) and
T3 (3.89 ± 0.2)
T2 = melatonin (p < 0.05). Sperm
implant alone progressive motility
and T3 = and kinematics,
control acrosomal and DNA
integrity improved
from T1 to T2 and T3
(p < 0.05)

Although the process by which PGF2α acts in the male is not well understood, it has a
potential impact on the hypothalamus-pituitary-testicular axis (Borg et al., 1992). Another
hypothesis is that in the boar, there is direct or indirect stimulation of some areas of the
nervous system, which would cause an increase in sexual appetite (Estienne et al., 2004).
Other recent studies have used synthetic PGF2α analogues in combination with Ox to make
semen collection by an electroejaculator more efficient and allow the collection of adequate
semen samples. For example, Ungerfeld et al. (2018) evaluated the combined administration
of 250 µg of cloprostenol and 10 IU of Ox (5 min and 30 s before the start of semen collection,
respectively) in male goats. Based on their results, the application of both hormones prior to
semen collection shortened the collection time and reduced the intensity of the electrical
stimulation required for semen sample collection, thus improving animal welfare without
affecting semen quality.
Notably, given the still limited use and lack of studies on the use of PGF2α in rams, further
studies are needed under different scenarios such as breeds, breeding seasons and doses,
especially determining whether it has a short half-life and a residual effect after its application for
certain periods such as days, weeks or months (personal communication, Ungerfeld et al., 2023).

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Table 3. Summary of the studies on PGF2α analogues, including breeds, doses, method of
administration, time of the study and the main effects.
Collection
Breeding Application Collection
Reference Breed time after Dose Main effects
season route method
application
Increased semen
volume, spermatozoa
Masoumi et al. Not Holstein 250 mg
IM Artificial vagina 30 minutes concentration and
(2011) reported bulls cloprostenol
testosterone level in
low-libido bulls
7.5 mg
cloprostenol Increased individual
24 hours motility and sperm
Maatoq et al. 5 buck
Within IM Artificial vagina 24 hours before semen concentration,
(2015) goats
collection for a decreased abnormal
period of sperm
5 weeks
30 minutes
Increased semen
before
20 volume, sperm
semen
Olfati et al. Crossbred 7.5 mg concentration, total
Outside IM Artificial vagina collection
(2013) rams with cloprostenol number of
twice a
Merino spermatozoa per
week/5
ejaculate
week
5 minutes 10 mg No positive effects on
Ungerfeld et al. 26 Saint
Within IM Does no apply before dinoprost sexual behaviour in
(2020) Croix rams
sexual tests tromtehamine rams
Animals in the
dinoprost-treated
group mated more
5 minutes 10 mg
times than those
before dinoprost
treated with
sexual tests tromtehamine
cloprostenol.
Sánchez- 24 Cloprostenol-treated
Davila et al. Within Katahdin IM Artificial vagina animals had a higher
(2022) rams mating efficiency
(ejaculations/total
20 minutes matings). Cloprostenol-
0.15 mg
before treated animals
cloprostenol
semen produced more semen
dextrogy
collection with better mass
motility

Melatonin
Melatonin (5-methoxy-N-acetyltryptamine) is one of the most studied and used hormones in goat
reproduction worldwide. It was first described in the bovine pineal gland in 1958 (Wurtman, 1985). In
the early 1980s, scientists started to elucidate how melatonin is regulated by the photoperiod and its
effect on testicular activity in rams (Almeida and Lincoln, 1984; Yu and Reiter, 1993). During this
decade, research focused on the effect of the photoperiod when rams were exposed to artificial light
cycles of short and long days, with most studies reporting a circadian mechanism and its correlation
with melatonin blood concentrations, photoperiod and reproductive status of rams (Lincoln et al.,
1981). In these studies, it was hypothesised that the response of rams to a change in day length
depends on ocular photoreception and is dictated by the combined activities of two brain areas, the
suprachiasmatic nucleus (SCN) and the pineal gland. At that time, the function of melatonin action in
ram reproduction started to be described (Lincoln et al., 1981; Ebling et al., 1988; Hanif and Williams,
1991; Fitzgerald and Stellflug, 1991) (see Figure 3).
Subsequently, in the 1990s, a line of research began to investigate the use of melatonin
implants in both sheep and rams due to the discovery of the action of melatonin through the
photoperiod (Fitzgerald and Stellflug, 1991; Webster et al., 1991; Lincoln and Maeda, 1992),
where rams treated with melatonin implants during a long photoperiod showed stimulated
testicular development through a pathway similar to that exerted by endogenous melatonin

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through the hypothalamus. From this point on, a cascade of investigations was developed
regarding the main factors that could contribute to an interaction with melatonin implants,
such as breed, diet, dose and implantation period, including the evaluation of semen quality
and sexual behaviour in rams (Hanif and Williams, 1991; Rosa et al., 2000).
In the following decade (2000–2010), the presence of melatonin in seminal plasma and its
antioxidant effects were found to be highly correlated with the activity of the three antioxidant
enzymes superoxide dismutase, glutathione reductase and catalase (Casao et al., 2010a). More
recent studies (from 2011 to date) have demonstrated direct effects of melatonin on ram
spermatozoa, reducing apoptotic effects and modulating sperm capacitation to improve
fertilisation rates (Shayestehyekta et al., 2022; Kumar et al., 2022). This was the guideline for the
development of studies on the addition of different concentrations of melatonin to diluted
semen to improve its quality in the freeze-thaw process since free radicals (ROS) are formed at
this stage (Rizkallah et al., 2022), which have an oxidative effect when they accumulate
excessively, altering the sperm cell membrane, resulting in a reduction in sperm motility and
viability. This can be explained by the fact that spermatozoa are rich in unsaturated fatty acids
and lose most of their cytoplasm during maturation, exposing them to the effects of ROS
(Moradi et al., 2020). In addition, more targeted studies have examined the effect of melatonin
on testicular haemodynamics (blood flow), which is important for providing oxygen and nutrients
to the testes and for eliminating metabolic waste (Shahat et al., 2022a; El-Shalofy et al., 2022).
Originally thought to be secreted only in the pineal gland, we now know that melatonin is
produced in other tissues throughout the body, particularly in the male reproductive tract
(Webster et al., 1991). As a multifunctional hormone, it is involved in various physiological
processes in mammals, such as cardiovascular regulation, immunomodulation, circadian
rhythms and, in some species, the regulation of the reproductive season (Gonzalez-Arto et al.,
2017; Abecia et al., 2020). It is one of the key hormones in sheep reproduction (Gonzalez-
Arto et al., 2017) and secreted in greater amounts by the pineal gland during long nights
(Figure 3) (Abecia et al., 2020; Jiang et al., 2022). This hormone sends signals that regulate the
seasonality of reproduction; in male sheep, it exerts specific effects on the hypothalamus,
triggering, by positive feedback, the pulsatile release of gonadotropins, thus establishing
control over sexual activity (Tsantarliotou et al., 2008; Harman and Serpek, 2022).
Melatonin has two mechanisms of action: 1) receptor-mediated action, namely the control
of seasonal reproduction and modulation of the sleep cycle, involving cAMP and/or
phospholipase C as cellular second messengers (Guerrero et al., 2007; Reiter et al., 2009).
Therefore, melatonin acts on the control of GnRH secretion and, consequently, on LH and FSH
release, affecting testicular activity and T release (Figure 3) (Reiter et al., 2007; Bronson, 2009;
Yu et al., 2018), where T secretion is mainly dependent on cAMP signalling, which is stimulated
by LH (Gonzalez-Arto et al., 2016). When Leydig cells are exposed to melatonin, T release and
cAMP production are increased depending on the dose of melatonin used (Li and Zhou, 2015);
2) receptor independence, which is a characteristic of melatonin due to its liposoluble and
water-soluble molecular nature, allowing it to reach all subcellular compartments at
physiological concentrations. Based on the above, when exogenous melatonin is administered,
high concentrations are reached in the cell membrane, in the nucleus and in the mitochondria.
Due to its indole chemical structure and high redox potential, melatonin can neutralise free
radicals, thereby reducing oxidative stress and decreasing sepsis toxicity (Reiter et al., 2009).
Melatonin secreted in the pineal gland acts in the regulation of reproductive seasonality
(Guerrero et al., 2007), having stimulatory effects in rams and male goats, which show
reproductive activity in the autumn (Tsantarliotou et al., 2008; Reiter et al., 2009). The
nocturnal secretion of melatonin in response to changes in the photoperiod is the passive
message for the hypothalamic-pituitary-gonadal axis to identify which period of the year it is
in (Harman and Serpek, 2022). The photoperiod is the common signal that determines the
change of seasons in mammals from higher latitudes to the mid-tropics (Bronson, 2009). To
date, melatonin receptors have been identified in neurons of the hypothalamus, in the pituitary
gland, and in the testes and accessory sex glands (Gonzalez-Arto et al., 2017; Abecia et al.,
2020; Yu et al., 2018). In the hypothalamus, where reproductive activity is modulated, the
density of melatonin receptors may determine the sensitivity to changes in the photoperiod.

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Review of the use of hormones in rams

Thus, species with numerous melatonin receptors in the central nervous system may use this
neurohormone as a cue to circadian and photoperiodic rhythms, whereas species with lower
numbers of receptors may use other cues to activate reproduction (Witt-Enderby et al., 2003).
To date, research on the benefits of using melatonin, either in the form of implants (Pool et al.,
2020a, b) or added to chilled or frozen semen samples, has focused on reducing the oxidative stress
induced by an excess of reactive oxygen species (ROS) in the seminal fluid (Ma et al., 2021; Casao et al.,
2010a These ROS are generated mainly as a by-product of chemical reactions of spermatogenic
processes, heat stress and the reproductive season (Casao et al., 2010b; Ofosu et al., 2021); such
reactions cause oxidative damage to the germ cells, which is manifested by a decrease in blood flow,
high levels of ROS and, ultimately, a decrease in semen quality (Pool et al., 2021; Shahat et al., 2022b).
It is worth noting that melatonin is a ubiquitous molecule and found in the seminal plasma of rams,
especially during the day, suggesting that it is secreted extrapineally by the reproductive tract
(Gonzalez-Arto et al., 2016), considering that MT1 and MT2 receptors have been found mainly in the
testis and seminal vesicle (Casao et al., 2010a). In this sense, when melatonin is used in the extender
to chill and freeze semen samples in rams, sperm capacitation (Casao et al., 2010b) occurs with the
addition of 1 µM melatonin, which causes a decrease in protein tyrosine phosphorylation and results
in low levels of ROS and AMPc (Gimeno-Martos et al., 2019). However, in a more recent study by
Pool et al., 2021, evaluating different levels of melatonin in ram semen, melatonin neither reduced
the intracellular ROS levels after semen thawing nor lipid peroxidation, but it did reduce mitochondrial
superoxide production and improved the motility and DNA integrity of frozen and thawed sperm
(Medrano et al., 2017). In another study, 18-mg melatonin implants were able to increase the activity
of the enzyme acrosin, which is important for initiating the acrosome reaction in the fertilisation
process (Kokolis et al., 2000).
A second melatonin application is in the form of implants impregnated with melatonin, the
use of which has been intensified both in sheep (Quintero et al., 2001; Ungerfeld, 2016) and
rams (Pool et al., 2021) (Table 4). As sheep are a seasonal polyoestrous species, their
reproductive activity is regulated by the photoperiod (Orihuela Trujillo, 2012; Leyva-
Corona et al., 2023), and information about the photoperiod is transmitted to the gonadal
system through the secretion of melatonin by the pineal gland. In rams, such implants can
contain 18 mg of melatonin, and in one study with an implantation period of 14 weeks, the T
levels increased until the 6th week post-implantation, also affecting testicular size and the
number of sperm per ejaculate, but not sperm motility and morphology (Pool et al., 2020b).

Table 4. Summary of the studies on melatonin, including breeds, doses, method of administration, time
of the study and the main effects.

Collection
Breeding Application Collection
Reference Breed time after Concentration Main effects
season method method
application
After melatonin
administration, emphasis
was on the secretory
epithelium
characteristics. Induced a
significant increase in the
number and height of
Silastic sheet principal cells that
Seminal (500-1DOw showed signs of
Palacín et al. Subcutaneous vesicles Corning increased secretory
Outside 8 Soay 88 days
(2008) implant were sheeting, activity. Main cytological
removed Midland, MI, alterations in the
USA) 1 g principal cells: increase in
the concentrically
arranged membranes of
secretory vacuoles and
glycogen granules,
appearance of numerous
lysosomes and
multivesicular bodies

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Review of the use of hormones in rams

Table 4. Continued…
Collection
Breeding Application Collection
Reference Breed time after Concentration Main effects
season method method
application
3, 18-mg Increased the percentage
melatonin of progressive motile
Subcutaneous Artificial implants spermatozoa
Outside 2 Aragonesa 66 to day 126
implant vagina (Melovine; Ceva
Sanidad Animal,
Casao et al. Barcelona, Spain)
(2010a) 3, 18-mg Increase in the scrotal
melatonin circumference without
16 Dohne Subcutaneous Artificial implants changing the seminal
Outside 30 to day 78
Merino implant vagina (Melovine; Ceva quality parameters of
Sanidad Animal, fresh and frozen/thawed
Barcelona, Spain) semen
Increased sperm
3, 18-mg
concentration at the end
melatonin
7 Barbarine of the experimental
Rekik et al. Subcutaneous Artificial implants
Outside 10 Saint 24 days period. Scrotal
(2015) implant vagina (Melovine; Ceva
Croix rams circumference increased,
Sanidad Animal,
and mating behaviour
Barcelona, Spain)
improved
Treated rams exhibited
certain sexual behaviours
and had higher plasma
3, 18-mg testosterone
melatonin concentrations
Abecia et al. Subcutaneous implants More of the ewes that
Outside 6 Aragonesa Blood 0-30 days
(2018) implant (Melovine; Ceva mated with those rams
Sanidad Animal, became pregnant and
Barcelona, Spain) produced more lambs
per ewe compared to
those that mated with
non-treated rams
Increased blood plasma
testosterone levels.
Growth in testicular size
and sperm count per
ejaculation between
31, Merino
Pool et al. Subcutaneous weeks 3 and 12 after
Outside and Poll Blood 0 to day 72 Unmentioned
(2020a) implant implantation. Reduced
Dorset
sperm DNA
fragmentation in Poll
Dorset rams during
multiple weeks of the
non-reproductive season
Melatonin administration
stimulated dendritic cells
Silastic sheet
and macrophages
Seminal (500-1DOw
Abd- Sub- through increasing the
vesicles Corning
Elhafeez et al Outside 8 Soay cutaneous 66 days size and number of the
were sheeting,
. (2021) implant endosomal
removed Midland, MI,
compartments, which
USA) 1 g
may correlate to
increased immunity
Induced improvements
in ejaculate volume,
3, 18-mg
colour and appearance,
6 Touabire x melatonin
Sub- sperm production and
Assani et al. Ladoum and Electroejac implants
Outside cutaneous 60 to day 135 morphological
(2023) Touabire x ulator (Melovine; Ceva
implant abnormality rate; sperm
Peul Peul Sanidad Animal,
motility and survival rate
Barcelona, Spain)
of spermatozoa were
significantly improved

Anim Reprod. 2024;21(2):e20240007 18/29

Review of the use of hormones in rams

However, in a recent study using the same type of melatonin implant in both females and
males, the group that had received the implant had significantly lower values for first oestrus,
conception rate and lambing compared to the control animals (Tölü et al., 2022).
In another study carried out by Abecia et al., 2018, in rams, where a light programme was
combined with melatonin implants, the rams that had received the light treatment plus the
placement of three melatonin implants still presented the highest levels of plasma T at 15 and
30 days after exposure to the natural photoperiod; the ewes introduced to the melatonin-
treated rams had a lambing percentage of 100% and a high fertility (1.44 ± 0.51 lambs/ewe)
compared to the control ewes (78% lambing and 1.00 ± 0.69 lambs/ewe). In the same direction,
but in young lambs, the use of implants with 18 mg of melatonin does not improve the sexual
behaviour of rams, but when exposed to a batch of ewes, the pregnancy rate was 89%; in
addition, treated lambs had better testicular development and higher T concentrations,
indicating that the use of melatonin implants in lambs can reduce the variability of age at sexual
maturity [98]. However, more recent studies have evaluated different doses of melatonin (18,
36 and 54 mg) in adult Border Leicester rams, and rams receiving 36- and 54-mg melatonin
implants showed an increase in scrotal circumference and T concentration, indicating the need
to use higher doses of melatonin and that the 36-mg dose is required to provoke an adequate
response of testicular size (Kleemann et al., 2021; Kleemann et al., 2022). In another study,
melatonin implants (36 mg) increased testicular volume, which is important in performing
testicular blood perfusion (TBP) studies for the diagnosis of reproductive disorders and in
assessing ram fertility (Akar et al., 2023). Table 4 shows a summary of some studies focussing
on the use of melatonin in the form of implants, summarising its positive effects on semen
quality and T concentration.

Conclusions and future implications

With the increasing demand for animal feed, the use of hormonal technologies to make
ram reproduction more efficient has accelerated in recent years. This implies that the
hormonal products mentioned in this review should always be accompanied by optimal animal
welfare measures and should not affect meat quality. For all hormonal products, there is a
wide variation in the length of time each has been evaluated and the impact it has on ram
reproduction. The reason for presenting six of the most important hormones used to improve
ram performance was to establish the current state of research on the effects of each of these
hormones on ram reproduction and to define their use in the immediate future, with a view to
achieving a balance between animal welfare and flock productivity on a global scale.

Acknowledgements

To the institutions of the authors involved in this review.

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Author contributions
EGB, KMT and FSD outlined the themes of the literature review and wrote the manuscript; EGB and KMT generated the figures; EGB, KMT, CCZC, CLP, JHM,
MGD, JFVA and FSD updated the references and revised the article. All authors have read and approved the final version of the manuscript.

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