Ram seminal plasma and its functional proteomic assessment.

Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa both in vitro and in vivo by affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and applied in vitro to prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.

The biological mechanisms regulating sperm selection by the ovine cervix.

In species where semen is deposited in the vagina, the cervix has the unique function of facilitating progress of spermatozoa towards the site of fertilisation while also preventing the ascending influx of pathogens from the vagina. For the majority of species, advances in assisted reproduction techniques facilitate the bypassing of the cervix and therefore its effect on the transit of processed spermatozoa has been largely overlooked. The exception is in sheep, as it is currently not possible to traverse the ovine cervix with an inseminating catheter due to its complex anatomy, and semen must be deposited at the external cervical os. This results in unacceptably low pregnancy rates when frozen-thawed or liquid stored (>24 h) semen is inseminated. The objective of this review is to discuss the biological mechanisms which regulate cervical sperm selection. We assess the effects of endogenous and exogenous hormones on cervical mucus composition and discuss how increased mucus production and flow during oestrus stimulates sperm rheotaxis along the crypts and folds of the cervix. Emerging results shedding light on the sperm-cervical mucus interaction as well as the dialogue between spermatozoa and the innate immune system are outlined. Finally, ewe breed differences in cervical function and the impact of semen processing on the success of fertilisation, as well as the most fruitful avenues of further investigation in this area are proposed.

D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

Penicillamine prevents ram sperm agglutination in media that support capacitation.

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 µM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

Variation in seminal plasma alters the ability of ram spermatozoa to survive cryopreservation.

Variation in the effect of seminal plasma on sperm function and fertility has been hypothesised to be due to differences between males and their seminal plasma composition. The freezing resilience of individual rams (n=17) was investigated to characterise inter-male variation. This was determined by measuring the degree of change in motility induced by cryopreservation (Experiment 1). Experiment 2 examined the effect of pooled seminal plasma from rams identified as having high or low resilience to freezing on the cryosurvival of washed spermatozoa from either high (n=3) or low (n=3) sperm freezing resilience rams. Immediately after thawing and throughout the incubation period (0-4h), spermatozoa from high-resilience rams frozen with high-resilience seminal plasma demonstrated superior motility to spermatozoa from high-resilience rams frozen with low-resilience seminal plasma (P<0.001). Similarly, spermatozoa from low-resilience rams frozen with high-resilience seminal plasma exhibited higher motility than spermatozoa from low-resilience rams frozen with low-resilience seminal plasma immediately after thawing (0h; P<0.001). The present study shows that variation in freezing resilience of ram spermatozoa is related to the source and composition of the seminal plasma.

Effects of varying doses of ß-nerve growth factor on the timing of ovulation, plasma progesterone concentration and corpus luteum size in female alpacas (Vicugna pacos).

Ovulation in camelids is induced by the seminal plasma protein ovulation-inducing factor (OIF), recently identified as ß-nerve growth factor (ß-NGF). The present study measured the total protein concentration in alpaca seminal plasma using a bicinchoninic acid (BCA) protein quantification assay and found it to be 22.2±2.0mgmL(-1). To measure the effects of varying doses of ß-NGF on the incidence and timing of ovulation, corpus luteum (CL) size and plasma progesterone concentration, 24 female alpacas were synchronised and treated with either: (1) 1mL 0.9% saline (n=5); (2) 4µg buserelin (n=5); (3) 1mg ß-NGF protein (n=5); (4) 0.1mg ß-NGF (n=5); or (5) 0.01mg ß-NGF (n=4). Females were examined by transrectal ultrasonography at 1-2-h intervals between 20 and 45h after treatment or until ovulation occurred, as well as on Day 8 to observe the size of the CL, at which time blood was collected to measure plasma progesterone concentrations. Ovulation was detected in 0/5, 5/5, 5/5, 3/5 and 0/4 female alpacas treated with saline, buserelin, 1, 0.1 and 0.01mg ß-NGF, respectively. Mean ovulation interval (P=0.76), CL diameter (P=0.96) and plasma progesterone concentration (P=0.96) did not differ between treatments. Mean ovulation interval overall was 26.2±1.0h. In conclusion, buserelin and 1mg ß-NGF are equally effective at inducing ovulation in female alpacas, but at doses ≤0.1mg, ß-NGF is not a reliable method for the induction of ovulation.

The Effect of Sperm Concentration and Storage Vessel on Quercetin-Supplemented Rabbit Semen During Chilled Storage.

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris-based extender supplemented with 100 µm quercetin to a concentration of 15, 30 or 60 × 10(6)  spermatozoa/ml, packaged into plastic tubes or 0.5-ml straws and stored at 15°C. Sperm quality was assessed by computer-assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2 O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10(6)  spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD-DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10(6)  spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10(6)  spermatozoa/ml may provide a suitable balance between motility and H2 O2 production to best maintain overall sperm function and should be evaluated in a large-scale AI trial.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Uterine tone influences fertility of Merino ewes following laparoscopic artificial insemination.

Artificial insemination (AI) plays a critical role in facilitating rapid genetic and production gains within the sheep industry. However, variable rates of AI success remain a concern for the industry and a barrier to adoption. Furthermore, the degree to which female factors influence the success of intrauterine laparoscopic AI rather than natural mating remains unknown. As such, this study investigates the effect of several factors collected during the time of AI, on the success of intrauterine laparoscopic AI. Data was generously donated by artificial breeding companies and stud breeders during routine commercial AI operations. AI data was collected over 3 breeding seasons during commercial AI programs (N = 24 programs) using Merino ewes (N = 24,700). Sire ID (N = 253), time of AI following progesterone removal (approx. 43-59 h post removal), uterine tone and intra-abdominal fat (both scored 1-5) as well as age of the ewe were all recorded at the time of AI. Transcutaneous ultrasound subsequently determined pregnancy rate approximately 55 days post-AI. A multivariate regression analysis was performed and revealed pregnancy success to increase when semen was inseminated into a ewe with a uterine tone score of 4 or 5 (P < 0.001). The remaining factors fell short of significance within the multivariate model. An interclass coefficient variation matrix was also used to determine the proportion of variation contributed to AI success by random factors allocated in the model; site, sire, AI date and breeding season (45.99 %, 29.94 %, 15.15 % and 8.92 %, respectively). These results highlight the influence of uterine tone on ewe fertility following laparoscopic AI, but also that program location and the sire used can further modify this influence on pregnancy rate. These factors must now be considered in combination with semen factors per individual sire used during AI to ascertain the contribution of several factors to the success of laparoscopic AI in Australia.

Factors affecting the success of laparoscopic artificial insemination in sheep.

Successful artificial breeding underpins rapid genetic and production gains in animal agriculture. In sheep, artificial insemination with frozen semen is performed via intrauterine laparoscopy as frozen-thawed spermatozoa do not traverse the cervix in sufficient numbers for high fertility and transcervical insemination is anatomically impossible in most ewes. Historically, laparoscopic artificial insemination has always been considered reasonably successful, but recent anecdotal reports of poor fertility place it at risk of warning adoption. Understanding the male, female and environmental factors that influence the fertility of sheep is warranted if the success of artificial insemination is to be improved and genetic progress maximised for the sheep industry. This review details the current practice of laparoscopic AI in sheep. It explores the effects of semen quantity and quality, the ewe, her preparation, and environmental conditions, on the fertility obtained following laparoscopic artificial insemination.

Birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa.

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

Seminal plasma and its effect on ruminant spermatozoa during processing.

Seminal plasma can both inhibit and stimulate sperm function, making its use as a supportive medium somewhat contradictory. These effects are directed by the multifunctional action of numerous inorganic and organic components, but it is the direct association of seminal plasma proteins with the sperm membrane that is thought to exert the most significant response. In vitro handling of spermatozoa in preparation for artificial insemination may involve washing, dilution, cooling, freezing, re-warming and sex-sorting. These processes can alter proteins of the sperm surface and reduce seminal plasma in the sperm environment. This, among other factors, may destabilize the sperm membrane and reduce the fertilizable lifespan of spermatozoa. Such handling-induced damage may be prevented or reversed through supplementation of seminal plasma, but the effectiveness of this technique differs with species, and the source and subsequent treatment of both spermatozoa and seminal plasma. Seminal plasma appears to act as a protective medium during in vitro processing of ram spermatozoa, but this does not appear to be the case for bull spermatozoa. The reasons for this divergent effect will be discussed with particular emphasis on the influence of the major proteins of ruminant seminal plasma, known as BSP proteins. The biochemical and biophysical properties of these proteins are well documented, and this information has provided greater insight into the signalling pathways of capacitation and the protective action of extender components.

Melatonin improves the motility and DNA integrity of frozen-thawed ram spermatozoa likely via suppression of mitochondrial superoxide production.

The ability of the neurohormone melatonin to ameliorate cryopreservation-induced damage to spermatozoa has been demonstrated in several domestic species. However, it is unclear how these protective effects are conferred, with improvements in sperm quality ambiguously attributed to the general antioxidant activity of melatonin. To further investigate this phenomenon, ram spermatozoa were diluted in cryomedia with and without melatonin (0 [control], 0.1, 1, 10, and 100 µM) and assessed for motility, viability, DNA integrity, mitochondrial superoxide production, lipid peroxidation, and intracellular reactive oxygen species before freezing and after thawing (0, 3, and 6 h post-thaw). Before freezing, supplementation with melatonin at any concentration had no effect on any measure of sperm quality. However, post-thaw, spermatozoa frozen in the presence of any level of melatonin reduced mitochondrial superoxide production of spermatozoa (P < 0.001), decreased the level of sperm DNA fragmentation (P < 0.001), and increased the percentage of motile spermatozoa (P = 0.035). Melatonin supplementation did not influence the relative levels of lipid peroxidation in the sperm membrane, the levels of intracellular reactive oxygen species, or sperm membrane lipid disorder (P > 0.05). There was no difference in the percentage of viable spermatozoa between treatment groups pre- or post-freeze (P > 0.05). These results suggest that, in the ram, melatonin does not protect the quality of cryopreserved spermatozoa through a nondiscerning scavenging of reactive oxygen species as previously suggested. Rather, melatonin appears to specifically reduce mitochondrial superoxide production, altering sperm functionality, as opposed to merely increasing the percentage of live sperm.

Sperm surface changes and their consequences for sperm transit through the female reproductive tract.

Spermatozoa are faced with considerable challenges during their passage through the female reproductive tract. Following deposition, they must deal with several physical and biochemical barriers as well as an aggressive immune defence system before they reach the site of fertilisation. While many factors are at play, the surface characteristics of spermatozoa are central to communication with the female and successful transit. The surface proteome of spermatozoa has been extensively studied and shown to vary considerably between species that deposit semen in the vagina (ram and bull) and uterus (boar and stallion), likely due to major differences in accessory sex gland anatomy. Comparing the surface characteristics of spermatozoa from these domestic species and how individual components may equip spermatozoa to interact with different features of the female tract could help understand how spermatozoa navigate from vagina or uterus to oviduct ampulla. Furthermore, we can begin to explain why use of high quality preserved spermatozoa in artificial insemination programs may still result in reduced fertility due to altered interaction with the female. In this review, we describe the sperm surface characteristics of the ram, bull, boar and stallion and compare changes as a result of mixture with seminal plasma and/or in vitro processing. The role of these seminal components in facilitating sperm survival and transit within the female reproductive tract is summarised, drawing attention to potential implications for applied reproductive technologies.

Overcoming neuroendocrine and metabolic barriers to puberty: the role of melatonin in advancing puberty in ewe lambs.

Pubertal onset in the ewe is subject to a multitude of physiological and environmental constraints. As seasonal breeders, sheep rely on decreasing photoperiod to enter puberty and the subsequent breeding periods, hindering production. The initiation of puberty defines the reproductive yield of the ewe, and as such is a critical factor influencing production outcomes. Currently, the misconception that ovine puberty is reliant on age results in ewes being bred at over a year old, leading to a substantial unproductive period between birth and first conception. As such, transcending pubertal barriers to allow for earlier initiation of reproductive competency has significant commercial merit. The primary candidate to achieve this is the neurohormone melatonin, a key factor that naturally signals photoperiodic change that facilitates seasonal remodeling of the ovine hypothalamic-hypophyseal-gonadal axis. Despite being known to modulate reproductive seasonality in both the mature ewe and ram, the ability of melatonin to advance ewe puberty remains underutilized in industry. To optimize melatonin application and shape perceptions of breeding ewe lambs, a greater understanding of pubertal impediments and the natural role of melatonin is warranted. This review examines the physiological role and applications of melatonin to advance ewe puberty, and how this may act in conjunction with other physiological and metabolic cues.

Treatment of rams with melatonin implants in the non-breeding season improves post-thaw sperm progressive motility and DNA integrity.

In the Merino ram, it is unclear whether cryopreserved sperm function and fertility is compromised when collected during the non-breeding season, when Merino ewes are seasonally anestrus. It was therefore investigated whether treatment with melatonin could improve sperm function or fertility when semen was collected during the period Merino ewes were seasonally anestrus. There were 16 Merino rams treated or not treated with melatonin implants during the non-breeding season of ewes (September). Ejaculates were collected before melatonin treatment (Week 0), during the period of melatonin release (Week 7) and subsequent breeding season (Week 23). In vitro sperm function was assessed before freezing, and at 0- and 3 -hs post-thaw. Fertility was determined through intrauterine insemination of ewes (n = 966) with frozen-thawed samples, during the breeding season. Compared to Week 0 values, spermatozoa from melatonin-treated rams had greater progressive motility at Week 7 (P = 0.019) and less DNA fragmentation (P = 0.003) at Weeks 7 and 23, whilst spermatozoa from non-treated rams were unchanged during these time-periods. There were no other treatment effects on sperm function or fertility (P > 0.05). In ejaculates collected during Week 23, there were no effects of treatment either before freezing or post-thawing. Sperm from ejaculates collected at Week 23, however, had lesser pre-freezing/post-thawing total motility and resulted in lower pregnancy rates (P < 0.05). It is concluded there are no effects of season on sperm quality or fertility of Merino rams and that melatonin treatment subtly improves quality of spermatozoa following cryopreservation.

Exogenous melatonin advances the ram breeding season and increases testicular function.

Governed by melatonin, ovine reproductive seasonality limits production outcomes due to periods of decreased reproductive efficiency. Though it is established that slow-release melatonin implants improve out of season reproductive performance in the ewe, the comprehensive effects of exogenous melatonin in the ram remain inconclusive. This study aimed to ultimately clarify the ability of exogenous melatonin to alter ram reproductive function during the non-breeding season and the subsequent breeding season. Hence, we investigated the effect of exogenous melatonin on reproductive endocrinology, semen quality and production, testicular size and libido in Merino and Poll Dorset rams (n = 31, using a subset of 18 rams for analysis of semen production and quality). Melatonin treatment resulted in elevation of melatonin in seminal plasma from 1-8 weeks post-implantation and in blood plasma at 6 weeks post-implantation. The blood plasma testosterone of implanted rams was greater than controls at both 6 weeks post-implantation and during the following breeding season. Implanted rams exhibited increased testicular size and number of sperm per ejaculate from 3-12 weeks post-implantation but did not demonstrate any change in sperm motility or morphology in response to treatment. Compared to their control counterparts, melatonin-treated Poll Dorset rams exhibited a lower percentage of sperm DNA fragmentation during several weeks of the non-breeding season. Though melatonin increased the likelihood of ejaculate collection in Poll Dorset rams (P < 0.05), libido was otherwise unaffected by treatment. Melatonin did not alter seminal plasma concentrations of inhibin A or Anti-Mullerian hormone, however, for the first time in the ram we have shown Anti-Mullerian hormone to be positively correlated with the number of sperm per ejaculate and sperm motility (r = 0.464 and 0.3242 respectively, P < 0.001), and inhibin A to be correlated to the number of sperm per ejaculate (r = 0.1786, P = 0.0135). These results indicate that melatonin is able to both systemically upregulate reproduction and act directly upon testicular function in the ram.

Sperm sexing in sheep and cattle: the exception and the rule.

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.

Germany/Australia index of sperm sex sortability in elephants and rhinoceros.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

Reproductive seasonality of male dromedary camels.

Reproductive seasonality has been reported in numerous species, including male dromedary camels, yet investigations into seasonal changes in camel semen quality have yet to be conducted. The aim of this study was to characterise the seasonal changes in camel semen quantity and quality as well as correlate these changes to testis and accessory sex gland morphology, sexual behaviour, libido and environmental factors such as day length and ambient temperature in Oman. Semen was collected twice a month for a year and testicular and accessory sex organ biometry recorded once a month via ultrasonography (n = 8 bulls). Blood samples were collected monthly to assess testosterone levels. Results indicated that testes and accessory sex glands size increased during October-April, peaking with testosterone concentrations during January (P<0.05). The sexual behaviour and libido of camels was also greater during the months of October-April (P<0.05). Attempts to collect semen were 100% successful during November-February. Semen volume, as well as sperm gross activity, concentration, motility, average path velocity and percentage with intact acrosomes were the greatest during January and decreased from May-September (P<0.05). Changes in values for semen variables, testosterone concentrations and sex organ anatomy were also highly correlated with seasonal changes in day length and ambient temperatures. In conclusion, a clearly defined reproductive season was observed in male camels in Oman ranging from December-March, with peak reproductive function occurring during December-January. To increase the success of breeding programs, matings or semen collections should be timed to occur when reproductive function is maximal.