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.

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.

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.

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.

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.

The fate of spermatozoa in the female reproductive tract: A comparative review.

The journey that spermatozoa take following deposition in the female tract is a long and perilous one. The barriers they face within the female tract differ depending on whether they are deposited in the vagina or uterus, like spermatozoa of the ram or boar respectively. Comparative studies on the transit of spermatozoa through the ewe and sow tracts serves to highlight similarities, or differences, in the way their sperm-surface properties enable them to overcome these barriers, progress through the tract and fertilise the oocyte. The female environment contributes towards this successful transit by providing a vehicle for sperm transport, aiding the removal of dead spermatozoa and other pathogens and applying strict selection pressures to ensure only those cells with the highest quality reach the site of fertilisation. Understanding the criteria behind these natural barriers helps an understanding of the limitations to fertility associated with preserved spermatozoa, and how in vitro manipulation can alter this complex interaction between spermatozoa and the female environment. Similar mechanisms or surface coat interactions exist in both species, but each has evolved to be used for physiologically disparate functions. Here we briefly describe the sperm surface characteristics of both fresh and frozen-thawed boar and ram spermatozoa and compare how these properties equip them to survive the physical, biochemical and immune interactions within the female reproductive tract.

Liquid storage of dromedary camel semen in different extenders.

This study was conducted to assess the effects of commercial extenders and storage temperature on dromedary camel sperm quality during liquid preservation. In Experiment 1, ejaculates (n = five males; replicated seven times) were split and diluted with synthetic (OPTIXcell, EquiPlus, INRA96, Bioxcell or AndroMed; Experiment 1a) or egg-yolk based (Biladyl, Green buffer or Triladyl; Experiment 1b) extenders and stored for 48 h at 4 °C. In Experiment 2, split ejaculates (n = five males; replicated six times) were used to directly compare Green buffer, OPTIXcell and Triladyl extenders over 48 h of storage at 4 °C. Ejaculates collected in Experiment 3 (n = five males; replicated five times) were diluted with Green buffer or Triladyl before chilled storage for 48 h at 4 or 15 °C. Sperm kinematics, viability and acrosome integrity were assessed during liquid storage. In Experiment 1a, there was the greatest total sperm motility (TM) in the OPTIXcell group following 24 and 48 h of storage, while in Experiment 1b, there was the greatest TM after 48 h of storage with Triladyl and Green buffer. In Experiment 2, there were greater TM and viable acrosome intact spermatozoa in the Triladyl and Green buffer than with OPTIXcell group. In Experiment 3, there was a greater TM in the Triladyl than Green buffer group at 24 and 48 h of storage regardless of storage temperature (which had no effect on sperm quality). In conclusion, camel sperm have greater viability when preserved in liquid form for 48 h following dilution with Triladyl and storage at either 4 or 15 °C.

Effect of semen collection frequency on the semen characteristics of dromedary camels.

The effect of semen collection frequency (once or twice per week) on the sexual behaviour, libido and semen characteristics (volume, colour, gross activity, viscosity, sperm concentration, morphology, motion characteristics and membrane viability and acrosome integrity) of dromedary camels (n = 7) was investigated over the course of 8 weeks. Results showed that frequency of collection influenced male camel libido (P < 0.05) but not sexual behaviour. Once per week collection frequency resulted in greater gross activity (2.7 ± 0.1 compared with 1.7 ± 0.1, P < 0.001) and greater sperm concentration (403 ± 16 compared with 261 ± 18 × 106 spermatozoa/mL, P < 0.001) compared to ejaculates collected twice per week. When collected twice per week, ejaculates collected during the first 3 weeks had a greater sperm concentration than those collected from week 4 onwards (P < 0.001). All ejaculates (100%) collected once per week 'qualified' (Criteria: > 60% total motility or > 100 × 106 spermatozoa/mL) for subsequent processing, but when collected twice per week the percentage of qualified ejaculates dropped sharply after three weeks (P < 0.001; 69% of ejaculates qualified over 8- week collection period). Twice weekly collection frequency caused a reduction (P < 0.001) in progressive motility, path velocity, track speed, lateral head amplitude, beat cross frequency and straightness. In conclusion, during the peak breeding season, semen can be collected twice per week from dromedary male camels for a period of 3 weeks only or once per week for 8 weeks without affecting semen quality.

Cryopreservation and egg yolk medium alter the proteome of ram spermatozoa.

Cryopreservation causes significant lethal and sub-lethal damage to spermatozoa. In order to improve freezing outcomes, a comprehensive understanding of sub-lethal damage is required. Cryopreservation induced changes to sperm proteins have been investigated in several species, but few have employed currently available state of the art, data independent acquisition mass spectrometry (MS) methods. We used the SWATH LC-MS method to quantitatively profile proteomic changes to ram spermatozoa following exposure to egg yolk and cryopreservation. Egg yolk contributed 15 proteins to spermatozoa, including vitellogenins, apolipoproteins and complement component C3. Cryopreservation significantly altered the abundance of 51 proteins. Overall, 27 proteins increased (e.g. SERPINB1, FER) and 24 proteins decreased (e.g. CCT subunits, CSNK1G2, TOM1L1) in frozen thawed ram spermatozoa, compared to fresh spermatozoa. Chaperones constituted 20% of the proteins lost from spermatozoa following cryopreservation. These alterations may interfere with both normal cellular functioning and the ability of frozen thawed spermatozoa to appropriately respond to stress. This is the first study to apply SWATH mass spectrometry techniques to characterise proteins contributed by egg yolk based freezing media and to profile cryopreservation induced proteomic changes to ram spermatozoa. SIGNIFICANCE: This study profiles changes to the sperm proteome induced by exposure to egg yolk based media and the process of cryopreservation, and the biological consequences are discussed.

The identification of proteomic markers of sperm freezing resilience in ram seminal plasma.

The source and composition of seminal plasma has previously been shown to alter the ability of spermatozoa to survive cryopreservation. In the present study, the ionic and proteomic composition of seminal plasma from rams with high (HSP; n = 3) or low (LSP; n = 3) freezing resilient spermatozoa was assessed. 75 proteins were identified to be more abundant in HSP and 48 proteins were identified to be more abundant in LSP. Individual seminal plasma proteomes were established for each of the six rams examined. For each ram, correlations were conducted between previously recorded freezing resilience [1] and individual spectral counts in order to identify markers of freezing resilience. 26S proteasome complex, acylamino acid releasing enzyme, alpha mannosidase class 2C, heat shock protein 90, tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase and transitional endoplasmic reticulum ATPase were found to be positively correlated (r(2) > 0.7) with freezing resilience. Cystatin, zinc-2-alpha glycoprotein, angiogenin-2-like protein, cartilage acidic protein-1, cathepsin B and ribonuclease 4 isoform 1 were found to be negatively correlated (r(2) > 0.7) with freezing resilience. Several negative markers were found to originate from the accessory sex glands, whereas many positive markers originated from spermatozoa and were part of or associated with the 26S proteasome or CCT complex.

Proteomic characterization and cross species comparison of mammalian seminal plasma.

Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE: This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.