Single Ejaculate Research Articles

D-Mannose-Mediated metabolic pathways sustain the molecular signatures of sperm function and fertilization.

Mammalian sperm within a single ejaculate exhibit significant heterogeneity, with only a subset possessing the molecular characteristics required for successful fertilization. Identifying the defining traits of these high-fertility sperm remains an open question. To elucidate the molecular markers and mechanisms underlying the fertilization potential of sperm in both mice and humans, with a focus on the role of D-mannose. Sperm morphology and functionality were analyzed using flow cytometry, biochemical assays, and immunofluorescence. Multi-omics analyses, including proteomics, metabolomics, and lipidomics, were conducted to identify distinct molecular signatures. Pharmacological interventions were employed to validate the role of key pathways, particularly Akt/mTOR signaling. Sperm with longer flagella demonstrated enhanced motility, mitochondrial activity, and fertilization potential in both mice and humans. Multi-omics analyses revealed distinct molecular profiles in high-fertility sperm, characterized by specific proteins, lipids, and metabolites. Notably, D-mannose supplementation enhanced sperm motility and fertilization capacity, even in asthenozoospermic sperm, by activating the Akt/mTOR pathway. This effect was not replicated by D-glucose or ATP supplementation. Mechanistically, D-mannose bypassed glycolytic rate-limiting steps, increasing ATP production and promoting mitochondrial and acrosomal integrity. This study identifies key molecular signatures of fertilization-competent sperm and highlights D-mannose as a novel modulator of sperm quality and function. These findings provide valuable insights into sperm biology and propose innovative therapeutic strategies for treating male infertility and optimizing assisted reproduction technologies.

O-285 Incompatible gametes: implications for infertility diagnosis and treatment

Abstract Sperm face intense selection in the female reproductive tract (FRT), and in humans usually only very few out of up to hundreds of millions of sperm cells can eventually proceed to the vicinity of an unfertilized oocyte. Traditionally, it has been thought that the primary function of female-induced sperm selection is to eliminate poor-quality sperm from reaching the oocyte. However, recent studies have demonstrated that in many species (including humans) FRT also mediate mate choice at the level of the gametes, which frequently bias fertilization towards the male, which is genetically compatible with the female. Furthermore, emerging evidence indicates that sperm phenotypic and genotypic variation within single ejaculates may allow gamete-level mate choice to act also among high-quality (‘healthy’) sperm and bias fertilization towards genetically compatible sperm haplotypes. I argue that both of these cellular-level mate choice processes can have important consequences for deeper understanding of the mechanistic basis of fertilization and infertility.

Effect of repeated semen ejaculation on sperm quality and selected biochemical markers of canine semen.

The aim of this study was to evaluate the quality parameters and selected biochemical markers of canine semen sampled at 24-h intervals over a period of 5 days, preceded by 6 months of sexual abstinence. Full ejaculates were obtained from 6 dogs. Ejaculate volume and total sperm counts in the ejaculate decreased gradually on successive sampling days. The percentage of total motile spermatozoa (TMOT), percentage of progressively motile spermatozoa (PMOT), sperm plasma membrane integrity (SPMI), and sperm mitochondrial membrane potential (MMP) increased on successive days of sampling. In addition, ATP content increased in spermatozoa. Total protein content (TPC) and the activity of aspartate aminotransferase (AAT), alkaline phosphatase (AP), and acid phosphatase (AcP) decreased in seminal plasma. Repeated ejaculation over a period of 5 days induced changes in the qualitative and quantitative parameters of canine semen. A decrease in the values of some biochemical markers of semen, secreted by the epididymis and the prostate gland, could point to disturbances in the secretory activity of these organs. Canine semen sampled after prolonged sexual abstinence is generally characterized by less desirable quality parameters, and this observation should be taken into consideration when semen is collected for artificial insemination or preservation. Semen quality can be significantly improved by repeating the sampling procedure after 24 hours. On the other hand, repeated sampling on successive days can significantly decrease total sperm counts in the ejaculate. As a result, a sufficient number of semen doses for artificial insemination may not be obtained from a single ejaculate.

QTLs and Candidate Genes Associated with Semen Traits in Merino Sheep.

Ram semen traits play a significant role in conception outcomes, which in turn may influence reproductive efficiency and the overall productivity and profitability of sheep enterprises. Since hundreds of ewes may be inseminated from a single ejaculate, it is important to evaluate semen quality prior to use in sheep breeding programs. Given that semen traits have been found to be heritable, genetic variation likely contributes to the variability observed in these traits. Identifying such genetic variants could provide novel insights into the molecular mechanisms underlying variability in semen traits. Therefore, this study aimed to identify quantitative trait loci (QTLs) associated with semen traits in Merino sheep. A genome-wide association study (GWAS) was undertaken using 4506 semen collection records from 246 Merino rams collected between January 2002 and May 2021. The R package RepeatABEL was used to perform a GWAS for semen volume, gross motility, concentration, and percent post-thaw motility. A total of 35 QTLs, located on 16 Ovis aries autosomes (OARs), were significantly associated with either of the four semen traits in this study. A total of 89, 95, 33, and 73 candidate genes were identified, via modified Bonferroni, within the QTLs significantly associated with volume, gross motility, concentration, and percent post-thaw motility, respectively. Among the candidate genes identified, SORD, SH2B1, and NT5E have been previously described to significantly influence spermatogenesis, spermatozoal motility, and high percent post-thaw motility, respectively. Several candidate genes identified could potentially influence ram semen traits based on existing evidence in the literature. As such, validation of these putative candidates may offer the potential to develop future strategies to improve sheep reproductive efficiency. Furthermore, Merino ram semen traits are lowly heritable (0.071-0.139), and thus may be improved by selective breeding.

Sperm vitrification supports in vitro blastocyst development after ICSI in horses

In the last decade, the introduction of advanced assisted reproductive techniques has significantly impacted the horse breeding industry. Specifically, in vitro embryo production by intracytoplasmic sperm injection (ICSI) has gained relevance and begun to be used to conserve wild equids. Kinetic sperm vitrification permits cell cryopreservation without permeating cryoprotective agents, facilitating the international commercialization and transportation of samples. This study aimed to assess embryo morpho-kinetic dynamics up to the blastocyst stage of ICSI embryos produced by injection of frozen (F) or vitrified (V) spermatozoa into horse eggs. Semen collection, as well as sperm vitrification and analysis were done as described by Consuegra et al. (Animal Reproduction Sciences. 2019; 206:69-77). Computer-assisted sperm analysis (Sperm Class Analyzer® CASA) and flow cytometry with double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI (propidium iodide) were performed to assess acrosomal integrity and sperm viability respectively. Frozen and vitrified sperm cells from a single ejaculate of a horse and a donkey were used. Oocyte collection, in vitro maturation, ICSI, time-lapse imaging andembryo culture were performed as reported by Bragulat et. al. (Theriogenology 2023; 195:199-208). Sperm analysis before cryopreservation showed: total motility (TMOT) 98.34%; progressive sperm motility (PMOT) 78.60%; intact-membrane sperm (IMS) 86.00%, and normal form (NF) 79.37%. After thawing, frozen sperm (F) analysis showed: TMOT 84.36%; PMOT 61.28%; IMS 77.50% and NF 86.21% and for vitrified sperm (V): TMOT 80.64%; PMOT 61.28%, IMS 81.00% and NF 68.97%. A total of 89 matured oocytes were injected. No significant differences were found in cleavage rates (F: 35/43, 81.39%; V: 35/46, 76.08%) or blastocyst rates at day 8 (F: 3/43, 6.97%; V: 8/46, 17.39%). Preliminary data from time-lapse imaging showed similar embryo morpho-kinetic dynamics between groups. Average hours after ICSI for time to cleavage were (mean ± SEM): F, n = 4, 27.00 ± 1.41; V, n = 3, 27.67 ± 1.20. Second cell division: F, n = 3, 38.33 ± 6.64; V, n = 3, 32.33 ± 3.38. Morula stage: F, n = 1, 85; V, n = 1, 120. Blastocyst stage: F, n = 1, 150; V, n = 1, 180. Our results demonstrate for the first time that vitrified sperm cells can be used successfully to produce blastocysts by ICSI without compromising developmental competence in horses.

Comparative efficacy of coconut water diluent with different semen extenders for cryopreservation of Nili Ravi buffalo bull semen

In this study, efforts were made to investigate fresh semen parameters and to select a suitable extender for buffalo semen cryopreservation. Experiment I, fresh undiluted seminal parameters were determined while Experiment II, efficacy comparison of coconut water extender (CWE) with tris citric acid extender (TCAE) and skimmed milk extender (SME) was made. Four bulls single ejaculate was collected weekly for 5 and 10 weeks for Experiment I and II, respectively however osmotic pressure replicates were 16 and 6 for semen and seminal plasma, respectively. In Experiment I, each bull spermatozoa concentration, motility (%), semen volume and pooled semen percentage NAR, PMI, viability and MTT (3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction rate was checked. In Experiment II, pooled semen added to extenders and then equilibrated (4oC) and filled to obtain 20×106 spermatozoa/0.5 ml straws before plunging in liquid nitrogen. Percentage thawed spermatozoa viability, normal acrosomal ridge (NAR), motility, DNA damage, plasma membrane integrity (PMI) and lipid peroxidation (nM) recorded. In Experiment I, seminal parameters as spermatozoa concentration (1226.43±71.48 million/mL), semen volume (2.84±0.14 mL), viability (90.05±0.71%), motility (77.13±0.71%), PMI (86.23±0.34%), NAR (94.67±0.30%) and MTT reduction rate (0.290±0.06) while osmotic pressure of seminal plasma (294.83±3.87 mOsm/kg) and semen (290.87±2.58 mOsm/kg) was recorded. In Experiment II, TCAE higher (P<0.05) sperm motility noted compared to CWE and SME whereas percentage viability, NAR, PMI, DNA damage was non-significant. Lipid peroxidation compared to SME and TCAE was higher (P<0.05) in CWE. In conclusion, based on sperm motility and lipid per oxidation, TCAE was more efficient for cryopreservation of buffalo semen.

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm.

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n=5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n=5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.

Cryptic female choice within individual males - A neglected component of the postmating sexual selection?

Cryptic female choice (CFC) is commonly assumed to act only in polyandrous mating systems, which allows females to bias fertilization towards the sperm of particular males. However, accumulated evidence has demonstrated that sperm show significant phenotypic and genotypic variation also within single ejaculates, which have important consequences for offspring phenotype and fitness. Here, I argue that these neglected sources of intra-male sperm variation often allow CFC to act also within individual males and facilitate fertilization bias towards genetically compatible (or otherwise preferred) sperm haplotypes. In this article, I explain prerequisites for within-male CFC, the criteria for demonstrating it and summarize accumulated evidence for this emerging selection process. Then, I evaluate prevalence of within-male CFC and review its potential evolutionary consequences. The aim of this article is to broaden the current definition of CFC by demonstrating that CFC has potential to act in all mating systems, in both internally and externally fertilizing species. Incorporation of the within-male CFC concept into the current models of sexual selection may provide novel insights into the deeper understanding of selective factors driving the evolution of mating systems and reproductive proteins. Finally, within-male CFC towards particular sperm haplotypes may increase our understanding of non-Mendelian inheritance.

Factors Affecting the Survival of Ram Spermatozoa during Liquid Storage and Options for Improvement.

Simple SummaryThe success of semen preservation is vital for the use of artificial reproductive technologies in sheep. However, reduced temperatures can cause significant damage to the sperm cell. Recent investigations in other species have identified room-temperature liquid storage as a viable alternative if spermatozoa are protected from the increased risk of lipid peroxidation, a side effect of unaltered metabolism. The following review aims to summarise the factors which contribute to the survival of ram spermatozoa during liquid storage and the role of pro-survival factors and antioxidants in helping to ameliorate the damaging effects caused by lipid peroxidation on fertility. This would contribute towards establishing a new method of semen preservation for the sheep industry which maximises fertility following storage and artificial insemination.Semen preservation is an essential component of reproductive technologies, as it promotes genetic gain and long-distance semen transport and multiplies the number of ewes able to be inseminated per single ejaculate. However, the reduced temperature during cold storage at 5 or 15 °C inflicts sub-lethal damage to spermatozoa, compromising sperm quality and the success of artificial breeding. New and emerging research in various species has reported the advantages of storing spermatozoa at higher temperatures, such as 23 °C; however, this topic has not been thoroughly investigated for ram spermatozoa. Despite the success of storing spermatozoa at 23 °C, sperm quality can be compromised by the damaging effects of lipid peroxidation, more commonly when metabolism is left unaltered during 23 °C storage. Additionally, given the biosafety concern surrounding the international transport of egg-yolk-containing extenders, further investigation is critical to assess the preservation ability of synthetic extenders and whether pro-survival factors could be supplemented to maximise sperm survival during storage at 23 °C.

STRAW SWIM UP PROCEDURE COMPARISON OF COMMERCIAL SPERM MEDIA EFFICACY

Our study aim is to compare the efficacy of commercial sperm processing media using the straw swim up method, since they have not been scrutinized to the same extent as embryo culture media. The Straw Swim Up procedure is an application of the conventional sperm swim-up technique, to easily analyze the ability of sperm to penetrate and migrate into the medium (Hecht and Jeyendran 1993).The procedure is simple and yields quantitative data. It is inexpensive and technically simple; multiple different media can be analyzed simultaneously using a single ejaculate; two independent sperm properties can be evaluated: migration into media and survivability in media; results are quantitative. Conventional methods of mixing semen with the test medium may fail to reveal the ability of sperm to penetrate and migrate into the test medium. Labeled 0.5ml straws (Reproduction Resources; Walworth, WI 53184, Catalog #440676), were filled by aspiration to 2 cm height individually with one of four different commercial media: Sperm washing Media (Fujifilm), (Fujifilm, Santa Ana, CA. 92705, Catalog #9983); Sperm Preparation Medium (Origio), (Origio, Trumbull, CT 06611, Catalog #10690060D); SAGE QUINN’S Sperm Washing Medium (Sage), (Cooper Surgical & Genomic Solutions, Denmark, Catalog #ART-1006); and Multipurpose Handling Medium with SSS (MHM), Fujifilm, Santa Ana, CA. 92705, Catalog #90163). These four straws were dipped into just below the surface of 18 different ejaculates and held vertically for 30 minutes. The sperm qualities recovered in the 0.5mL straws were statistically analyzed using ANOVA, and the mean and standard deviation (SD) of the results are listed in Table 1.Table 1Mean ± SD Sperm Quality Recovered in the 0.5mL Straws*Commercial MediumSperm Concentration x 106/mlMedian Sperm Concentration x 106/mlPercent Sperm Motility (%)Percent Sperm Progressive Motility (%)Fujifilm11.6 ± 7.99.077.7 ± 16.664.8 ± 18.7Origio9.1 ± 8.66.583.1 ± 15.069.4 ± 19.7Sage8.5 ± 5.39.378.7 ± 14.563.4 ± 16.2MHM11.2 ± 10.57.781.5 ± 12.265.3 ± 16.9*Mean ± SD of Sperm Concentration (98.5 ±24.7 x 106/ml), Sperm Motility (60.3 ± 13.0%) and Progressive Sperm Motility (40.7 ± 15.6%) of 18 ejaculates. Open table in a new tab *Mean ± SD of Sperm Concentration (98.5 ±24.7 x 106/ml), Sperm Motility (60.3 ± 13.0%) and Progressive Sperm Motility (40.7 ± 15.6%) of 18 ejaculates. There was no statistically significant difference in the quality of sperm recovered in the straws. However, an observable difference of the recovered sperm concentration median value among the four media was noted (Table 1). Future studies may confirm the differences in the response of the sperm from different ejaculates to penetrate and migration through the various sperm media.

Fertilization potential test of sperm from nano monoclonal antibody injected goats

• Over 90 % of X bearing sperm could be available using NMA™ sex selection technique. • In vitro fertility potential of sperm sorted by NMA™ injection was comparable to that in the control group. • The percentage of female offspring was more than 90 %. Controlling the sex of an animal’s offspring has long been the dream of most livestock producers. To produce a very higher percentage of X chromosome bearing sperm, calcium phosphate nano monoclonal antibody (NMA™) against Guanzhong dairy goat sperm epitope was developed in advance, and the detailed development technique has been applied for patent protection. The present study focused on the fertilization potential detection of sperm which was treated by testis injection of NMA™ to implementing sex selection of Guanzhong dairy goat. Results showed that no significant differences in sperm viability, sperm velocities, acrosomal integrity and plasma membrane integrity, although the volume and density of a single ejaculate were slightly lower in goats from the NMA™ injection group than those of the control group. From the 6th to 30th week following NMA™ injection, the percentage of X chromosome bearing sperm was more than 90 %; at the week 40th, it dropped to 88 %. After the second NMA™ injection performed at the 40th week, over 90 % of sperm with X chromosome could be obtained again. Moreover, the in vitro fertilization capacity of sperm derived from NMA™ injection was the same as that of the control group. No significant difference in pregnancy rates and kidding rates was observed following artificial insemination using sorted sperm versus unsorted sperm, but the proportion of female offspring was more than 90 %, which was significantly higher in the sex sorted group than that of the control group. The sex determination technique presented here can be mainly applied to facilitate the mass production of female Guanzhong dairy goats.

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics.

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1‐hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane‐intact spermatozoa determined by computer‐assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen‐thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.

Characteristics of semen collected from gander included in the genetic resources conservation program

Conservative breeding ex situ in vivo is one of the most popular methods of creating genetic reserves. Unfortunately, keeping animals in small closed populations leads to inbreeding which reduces their reproductive capacity. The aim of the study was to characterize the sperm quality of 6 genetic groups of geese (northern and southern breeds) kept in Poland for many generations as genetic reserve flocks. Each breed was represented by 10 randomly selected 1-yr-old ganders, semen was collected 14 times, individually from each male, and the number of positive reactions (ended with ejaculation), semen volume, sperm concentration, and morphology were assessed. The obtained results showed a significant difference between breeds and individuals of the same group, both in males’ reaction and semen quantitative and qualitative traits. From the northern breeds 193 ejaculates were obtained in total (i.e., 45.9% of all attempts), from the southern breeds 242 ejaculates (57.6%). The volume of single ejaculate varied from 0.01 mL (one drop allowing only histological smear and sperm morphology evaluation) to 0.65 mL; sperm concentration varied from 23.0 × 106mL−1 to 2376.0 × 106mL−1; the amount of total live sperm was at a similar level in all breeds (89.6%–97.7%), while live normal cells ranged between 15.2% and 67.9% depending on breed and individuals. When keeping the genetic reserves ex situ in vivo, attention should be paid to the quality of semen and males that are poor in this respect should be eliminated, in order not to lead to an excessive weakening of the reproductive capacity of the flocks covered by the genetic resources protection program.

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.

Sertoli cell proliferation in juvenile boars and microRNA

Sertoli cell numbers are a major influence on sperm production capacity. Increased use of artificial insemination and the limited number of insemination doses from a single boar ejaculate provide the incentive to increase Sertoli cell numbers for potential increases in sperm production and pig production efficiency. Although the number of Sertoli cells can be at least temporarily manipulated in vivo, the mechanism for the increased proliferation is poorly understood and critical for development of practical production practices. MicroRNAs are recognized to regulate many genes during development. Recent work suggested specific microRNAs were involved with regulation of Sertoli cell lines maintained in monoculture. In order to evaluate the relationship of specific microRNAs (miR-26a, miR-196a, miR-499, miR-638 and miR-1285) to in vivo Sertoli cell proliferation, three in vivo models of increased Sertoli cell proliferation (hemicastration, reduced androgen signaling, and reduced endogenous estrogen synthesis) were evaluated in young juvenile boars. Hemicastration was associated with slightly decreased miR-26a, similar to the decrease associated with in vitro Sertoli cell monoculture but none of the other microRNAs were affected by hemicastration nor were any affected by the blockade of androgen signaling with flutamide. Decreased expression of miR-638 followed reduced endogenous estrogen synthesis consistent with in vitro results although expression of miR-1285 was the converse from the in vitro results. Alterations in specific microRNAs are associated with increased Sertoli cell proliferation. However, altered expression of microRNAs was specific to the particular model system, suggesting unique molecular pathways are involved.

154 A method of oviductal semen deposition for use in the goat

The objective of this study was to investigate a method of oviducal semen deposition as a strategy for producing offspring from poor-quality cryopreserved goat sperm. Invitro fertilisation (IVF) and AI are common assisted reproductive technologies used in small ruminants, but they have varied results in the goat. The use of poor-quality cryopreserved-thawed sperm (&amp;lt;50% live/dead ratio at post-thaw) can decrease the rate of success. These procedures were performed in the month of November in Central Massachusetts in the United States (42° N). Seven 10-year-old dairy goats (Saanen, Toggenburg, and Alpine breeds) were synchronised and superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, two daily injections of 36mg of FSH ~12h apart on Days 12-15, and progesterone implant removal on Day 14 followed by an injection of 50µg of gonadotrophin-releasing hormone. Sperm deposition was performed on Day 17 (72 h after implant removal). The animals were anaesthetised using a standardised protocol, intubated, and maintained using isoflurane, and sterile prep was performed before a midline laparotomy procedure. Straws from a single ejaculate from a transgenic founder that was cryopreserved using a commercial two-step glycerol-egg yolk-based extender were used. A straw from this collection was post-thawed 30 days after collection and, using a commercial live/dead stain, 67% live sperm was determined. The optimal type of sperm prep and sperm concentration is unknown and may be dependent on sperm quality. Therefore, different gradient preps using Vitrolife SpermGrad at three volumes (1.5 (used on two animals), 1.0, and 0.5mL) as well as two volumes of IVF Bioscience Bovine BO-SemenPrep (4.0mL (used on two animals) and 2.0mL) were used. All five pellets were diluted in 1.0mL of IVF Bioscience Bovine BO-IVF media. Sperm concentrations ranging from 75×106 to 27×106 spermmL−1 were deposited into one oviduct; then, a 10:1 dilution was performed and 7.5×106 to 2.7×10 spermmL−1 were deposited into the contralateral oviduct. The depositions were performed just proximal to the uterotubal junction in a volume of 0.1mL of diluent via a tuberculin syringe attached to a 20-gauge needle. Two days following the procedure, oviducts were flushed postmortem from three of the seven randomly selected goats. All three had fertilised embryos, and nineteen 8-cell embryos were retrieved. Three of these embryos were surgically transferred to the distal uterine horn of a suitable recipient. The recipient became pregnant and produced a single offspring. The remaining four of seven goats were killed 41 days post-surgery. Two of the four goats were pregnant, with one carrying one fetus and the other carrying five fetuses. Further studies are needed to optimise this method, but these initial results indicate that oviducal semen deposition directly into the oviduct proximal to the uterotubal junction may be a suitable alternative for producing offspring from suboptimal cryopreserved-thawed goat sperm.

Study on the Effects of Multiple Matings in Coccinella transversalis for its Behaviour and Reproduction

The evolution and maintenance of multiple mating (repeated mating or polyandry) in predaceous ladybird beetles (Coleoptera: Coccinellidae) is an adaptive puzzle; since a single ejaculate of male often provides enough sperm to female for her lifetime egg production. Despite numerous studies on multiple mating evaluating reproductive attributes, there are negligible studies that investigated changes in behavioural patterns in ladybirds during multiple mating. In the present study, effects of multiple mating on mating behavioural pattern of males and reproductive attributes of females have been assessed using Coccinella transversalis as an experimental ladybird species. Results revealed that during copulation behaviour, time for the commencement of mating, latent period, wriggling movement duration, number of bouts and mating duration decreased with increase in number of mating; whereas interval between successive bouts increased significantly. Moreover, fecundity and egg viability of females increased with increase in number of mating.

Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose–EDTA–egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk–egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.

Evolutionary Proteomics Reveals Distinct Patterns of Complexity and Divergence between Lepidopteran Sperm Morphs.

Spermatozoa are one of the most strikingly diverse animal cell types. One poorly understood example of this diversity is sperm heteromorphism, where males produce multiple distinct morphs of sperm in a single ejaculate. Typically, only one morph is capable of fertilization and the function of the nonfertilizing morph, called parasperm, remains to be elucidated. Sperm heteromorphism has multiple independent origins, including Lepidoptera (moths and butterflies), where males produce a fertilizing eupyrene sperm and an apyrene parasperm, which lacks a nucleus and nuclear DNA. Here we report a comparative proteomic analysis of eupyrene and apyrene sperm between two distantly related lepidopteran species, the monarch butterfly (Danaus plexippus) and Carolina sphinx moth (Manduca sexta). In both species, we identified ∼700 sperm proteins, with half present in both morphs and the majority of the remainder observed only in eupyrene sperm. Apyrene sperm thus have a distinctly less complex proteome. Gene ontology (GO) analysis revealed proteins shared between morphs tend to be associated with canonical sperm cell structures (e.g., flagellum) and metabolism (e.g., ATP production). GO terms for morph-specific proteins broadly reflect known structural differences, but also suggest a role for apyrene sperm in modulating female neurobiology. Comparative analysis indicates that proteins shared between morphs are most conserved between species as components of sperm, whereas morph-specific proteins turn over more quickly, especially in apyrene sperm. The rapid divergence of apyrene sperm content is consistent with a relaxation of selective constraints associated with fertilization and karyogamy. On the other hand, parasperm generally exhibit greater evolutionary lability, and our observations may therefore reflect adaptive responses to shifting regimes of sexual selection.

Selection for longer lived sperm within ejaculate reduces reproductive ageing in offspring.

Males produce numerous sperm in a single ejaculate that greatly outnumber their potential egg targets. Recent studies found that phenotypic and genotypic variation among sperm in a single ejaculate of a male affects the fitness and performance of the resulting offspring. Specifically, within‐ejaculate sperm selection for sperm longevity increased the performance of the resulting offspring in several key life‐history traits in early life. Because increased early‐life reproductive performance often correlates with rapid ageing, it is possible that within‐ejaculate sperm selection increases early‐life fitness at the cost of accelerated senescence. Alternatively, within‐ejaculate sperm selection could improve offspring quality throughout the life cycle, including reduced age‐specific deterioration. We tested the two alternative hypotheses in an experimental setup using zebrafish Danio rerio. We found that within‐ejaculate sperm selection for sperm longevity reduced age‐specific deterioration of fecundity and offspring survival but had no effect on fertilization success in males. Remarkably, we found an opposing effect of within‐ejaculate sperm selection on female fecundity, where selection for sperm longevity resulted in increased early‐life performance followed by a slow decline, while females sired by unselected sperm started low but increased their fecundity with age. Intriguingly, within‐ejaculate sperm selection also reduced the age‐specific decline in fertilization success in females, suggesting that selection for sperm longevity improves at least some aspects of female reproductive ageing. These results demonstrate that within‐ejaculate variation in sperm phenotype contributes to individual variation in animal life histories in the two sexes and may have important implications for assisted fertilization programs in livestock and humans.