Sperm-rich Fraction Research Articles

Supplementation of glutamine in a short-term boar semen extender during 17°C holding time enhances post-thaw sperm quality for cryopreservation

BackgroundGlutamine is a nonessential amino acid and the most abundant amino acid found in the seminal plasma and sperm-rich fraction of boar semen. Glutamine plays an important role in enhancing glutathione (GSH) synthesis. It acts as an effective antioxidant in semen and provides intracellular defense to sperm against oxidative stress. This study aimed to improve the quality of frozen-thawed boar semen by using glutamine supplementation in a short-term semen extender during the holding time at 17 °C before cryopreservation.ResultsThe results indicate that the total motility, progressive motility, LIN, STR, and WOB were the highest in the 20 mM supplementation group at the 2 h timepoint after thawing. Thus, the optimal concentration for glutamine supplementation in short-term boar semen extender during the holding time at 17 °C was 20 mM. Interestingly, at all of the time points after thawing, 20 mM glutamine supplementation exhibited the highest level of sperm viability and membrane integrity when compared to the CONTROL (0 mM) and other experimental dilution groups. Moreover, the acrosome integrity, mitochondrial activity, and capacitation status (F pattern) were significantly greater in the 20 mM supplementation group than the other groups at the 2 h timepoint after thawing.ConclusionSupplementation of glutamine at a concentration of 20 mM in a short-term semen extender (Bio Pig®) during the 17 °C holding time before cryopreservation, which had a standard freezing extender (9.0% glycerol and 1.9% Equex paste), could enhance the post-thaw sperm motility and quality parameters of cryopreservation.

Evaluation of specific fractions of Large White Yorkshire boar semen

The current study was conducted to evaluate the quality of specific fractions of three Large White Yorkshire (LWY) boar semen samples. Fifteen semen samples were collected by gloved hand method as an initial 10 mL of sperm rich fraction (SRF- F1), remaining SRF (F2) and whole semen. Samples were evaluated for colour, volume, pH, sperm concentration and progressive motility. The first 10 mL of SRF of boar semen contained 25 per cent of the total ejaculate spermatozoa and was able to withstand cooling, freezing and thawing better than the spermatozoa of the bulk ejaculate. A significant difference between the fractions was observed in the volume of semen ejaculated, pH and sperm concentration across different fractions of the same ejaculate. The differences in semen fractions were attributed to the differences in the composition of seminal plasma of the initial fraction of SRF Keywords: Boar semen, sperm rich fraction, semen evaluation

Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The ‘sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma

BackgroundPorcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE).MethodsSeminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling.ResultsEach sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04–98.50%).ConclusionsThis study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

MicroRNA expression in specific segments of the pig periovulatory internal genital tract is differentially regulated by semen or by seminal plasma

microRNAs play pivotal roles during mammalian reproduction, including the cross-talk between gametes, embryos and the maternal genital tract. Mating induces changes in the expression of mRNA transcripts in the female, but whether miRNAs are involved remains to be elucidated. In the current study, we mapped 181 miRNAs in the porcine peri-ovulatory female reproductive tract: Cervix (Cvx), distal and proximal uterus (Dist-Ut, Prox-Ut), Utero-tubal-junction (UTJ), isthmus (Isth), ampulla (Amp), and infundibulum (Inf) when exposed to semen (natural mating (NM) or artificial insemination (AI-P1)) or to infusions of sperm-free seminal plasma (SP): the first 10 mL of the sperm rich fraction (SP-P1) or the entire ejaculate (SP-E). Among the most interesting findings, NM decreased mir-671, implicated in uterine development and pregnancy loss prior to embryo implantation, in Cvx, Dist-UT, Prox-UT, Isth, and Inf, while it increased in Amp. NM and SP-E induced the downregulation of miR-let7A-1 (Dist-UT, Prox-UT), a regulator of immunity during pregnancy. miR-34C-1, a regulator of endometrial receptivity gene expression, was increased in Dist-UT, UTJ and Amp (NM), in Prox-UT (AI-P1), and in Amp (SP-P1). miR-296, a modulator of the inflammatory response and apoptosis, was upregulated in the UTJ (all treatments). NM elicited the highest miRNA activity in the sperm reservoir (UTJ), suggesting that key-regulators such as miR-34c or miR-296 may modulate the metabolic processes linked to the adequate preparation for gamete encounter in the oviduct. Our results suggest that SP should be maintained in AI to warrant miRNA regulation within the female genital tract for reproductive success.

Characterization of the semen microbiota of healthy stud dogs using 16S RNA sequencing

The reproductive microbiota of male dogs has never been investigated using culture-independent sequencing techniques. The purpose of the present study was to get seminal knowledge on the microbiota of the ejaculate. Specifically, factors as the fraction of the ejaculate, the sperm quality (normospermia, teratozoospermia), and the living environment were evaluated. The sperm-rich and the prostatic fractions of the ejaculate were collected from healthy stud dogs. Following the sperm analysis, samples from twenty animals (normospermic n = 10 and teratozoospermic n = 10) were stored at – 80 °C until further processing including DNA extraction and 16S rRNA sequencing. Alpha- (Shannon index) and beta- (Bray-Curtis, Unweighted UniFrac) diversities were assessed and compared (PERMANOVA) based on the group of samples (biological samples from the ejaculate and controls), the fraction of the ejaculate (sperm-rich and prostatic fractions), the animal group (normospermia and teratozoospermia), and the living environment of the animal (kennel or pet living in-house). The most abundant bacterial phyla in canine semen samples were Proteobacteria, Firmicutes, and Actinobacteria. Overall, the dominant bacterial family was that of Pasteurellaceae The genus Mycoplasma was never detected. No differences in terms of bacterial composition were found based on the fraction of the ejaculate and based on the animal group (P > 0.05). On the other hand, differences in alpha and beta diversities were highlighted based on the living environment (P = 0.001).Overall, the results of the present study provide preliminary insights on dog semen microbiota, opening a new chapter in the field of canine andrology. Our results suggest that the environment may play a role in influencing the reproductive microbiota of male dogs and that the prostatic fraction of the ejaculate can be used for further research as a representative of the semen microbiota.

Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

BackgroundProtamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated.ResultsThis study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001).ConclusionWhile sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.

Different seminal ejaculated fractions in artificial insemination condition the protein cargo of oviductal and uterine extracellular vesicles in pig.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes.

Frozen Semen Quality of Kolbroek Boars Concerning Glutathione Concentrations

Glutathione(GSH) is an antioxidant that is being studied for its ability to improvecellular responses to semen preservation challenges. The research goal aimed toevaluate the impact of introducing different glutathioneconcentrations to boar semen freezing extender (0, 1, 5, and 10 mM) onthe Kolbroek boar sperm's quality after being frozen and thawed. For 2 h, thecollected sperm-rich fraction was chilled at 17°C. Following the 1 h ofequilibration, fraction B: Control (egg yolk 20% + BTS 72% + glycerol 8%), 1 mM(egg yolk 20% + BTS 70% + glycerol 8% + GSH 2%), 5 mM (egg yolk 20% + BTS 62 + 8%glycerol + GSH 10%) and 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%)was added into the semen samples. Following that, 0.5 mL straws with thediluted, cooled semen samples were submerged in liquid nitrogen (LN2)vapor and then stored inside the LN2 tank (-196°C). After thawing(37°C), parameters of boar sperm motility, velocity, morphology and viability,membrane permeability, and Malondialdehyde content were assessed and recorded.Using the analysis of variance, the data were examined. The results revealed adifference in total motility (%) on fresh semen as compared to post-thawedcontrol and GSH treatments. A significant difference was revealed in spermprogressive motility (%) in fresh semen (27.07±4.5). Significant differenceswere recorded in live sperm morphology and viability on fresh semen (81.8±2.8%)compared to control, 1, 5, and 10 mM. In the10 mM diluted semen (78.5±6.8), the proportion of sperm with an intact plasmamembrane integrity was highest (P<0.05), while the percentage in the1 mM diluted semen (73.0±2.7) was lower. Malondialdehyde levels were lowest (P<0.05)in the group receiving 5 mM treatment. In conclusion, 5 mM of GSH is therequired amount to be added to the freezing extender while cryopreserving semenfrom Kolbroek boars.

Proteomics Evaluation of Semen of Clinically Healthy Beagle-Breed Dogs.

The objectives of the present work were to evaluate the semen of dogs by means of proteomics methods and to compare with proteomics results of the blood of the animals, in order to increase available knowledge on the topic and present relevant reference values for semen samples. Semen samples were collected from five Beagle-breed dogs. Reproductive assessment of the animals by means of clinical, ultrasonographic and seminological examinations confirmed their reproductive health. The sperm-rich fraction and the prostatic fraction of semen were processed for proteomics evaluation. LC-MS/MS analysis was performed by means of a LTQ Orbitrap Elite system. The technology combines high separation capacity and strong qualitative ability of proteins in biological samples that require deep proteome coverage. Protein classification was performed based on their functional annotations using Gene Ontology (GO). In blood plasma, semen sperm-rich fraction, and semen prostatic fraction, 59, 42 and 43 proteins, respectively, were detected. Two proteins were identified simultaneously in plasma and the semen sperm-rich fraction, 11 proteins in plasma and the semen prostatic fraction, and three proteins in the semen sperm-rich and prostatic fractions. In semen samples, most proteins were related to cell organization and biogenesis, metabolic processes or transport of ions and molecules. Most proteins were located in the cell membrane, the cytosol or the nucleus. Finally, most proteins performed functions related to binding or enzyme regulation. There were no differences between the semen sperm-rich fraction and prostatic fractions in terms of the clustering of proteins. In conclusion, a baseline reference for proteins in the semen of Beagle-breed dogs is provided. These proteins are involved mostly in supporting spermatozoan maturation, survival and motility, enhancing the reproductive performance of male animals. There appears potential for the proteomics examination of semen to become a tool in semen evaluation. This analysis may potentially identify biomarkers for reproductive disorders. This can be particularly useful in stud animals, also given its advantage as a non-invasive method.

A retrospective study of the relationship between canine age, semen quality, chilled semen transit time and season and whelping rate and litter size

The objectives of this study were to investigate the effect of the dog’s age, semen quality, and the duration and season of semen transit on whelping rate and litter size after insemination with transported chilled extended semen. The sperm rich fraction was collected from 43 dogs of 18 breeds, which were presented at the Clinic for chilled semen transport, in the period from 2017 to 2021. Immediately after collection, the total sperm concentration and count, motility, membrane integrity (HOS test), the percentage of live spermatozoa and sperm morphology (eosin nigrosin staining) were evaluated. The sperm rich fraction was centrifuged and diluted with Tris – fructose - citrate extender with the addition of 20% (v/v) egg yolk, then chilled and prepared for shipping. A dose consisted of at least 200x106 live, motile, morphologically normal spermatozoa. The data on the dog’s age, chilled sperm transit time, the season of transit, and the whelping rate and litter size after insemination were recorded. The whelping rate was 55.8% with a mean (±SEM) litter size of 4.71±0.58 pups. The total number of spermatozoa was higher in artificial insemination (AI) that resulted in whelping compared to unsuccessful AI (P&lt;0.05). No difference was observed for any of other sperm quality parameters tested, such as the dog’s age or season of transit regarding whelping rate or litter size. Transit time significantly affected the whelping rate (P&lt;0.01), at (mean±SEM) 21.50±1.28 and 37.00±5.59 h in successful and unsuccessful AI, respectively. In conclusion, analysis of the factors related to the dogs identified total sperm count and transit time as factors that significantly affected whelping rates in bitches inseminated with transported chilled extended semen.

Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10–14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0–d5), T2 (d6–d10), and T3 (d11–d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Should All Fractions of the Boar Ejaculate Be Prepared for Insemination Rather Than Using the Sperm Rich Only?

Simple SummaryThe swine industry is constantly looking for efficiency improvement, especially focusing on the artificial insemination (AI) process. One of the trends in AI centers is to maximize the number of doses obtained from one ejaculate. Seminal doses are usually prepared with the sperm-rich fraction or the whole ejaculate, but further studies are needed to understand how to prepare them properly. Thus, this study aims to analyze how accumulative ejaculate fractions may influence sperm storage, AI performance, and offspring. The results indicate that the presence of all ejaculate fractions within seminal doses does not affect either sperm quality or AI performance and offspring health. Therefore, this study highlights the possibility to use the bulk ejaculate for seminal dose preparation, leading to successful AI. Additionally, it results in a more time-efficient preparation of a greater number of seminal doses providing an economic advantage.Boar ejaculate is released in several well-characterized fractions, differing in terms of sperm concentration, seminal plasma volume, and composition. However, the inclusion of the last part of the ejaculate for artificial insemination (AI) purposes is still under debate due to its controversial effects. Thus, there is a need to study the potential synergistic impact of the different ejaculate fractions. We aimed to evaluate the effect of accumulative ejaculate fractions on sperm conservation, AI performance, and offspring health. Ejaculates (n = 51) were collected and distributed as follows: F1: sperm-rich fraction; F2: sperm-rich + intermediate fractions; F3: sperm-rich + intermediate + poor fractions. Each group was diluted in a commercial extender, packaged in seminal doses (2000 × 106 sperm/60 mL), and stored at ~16 °C. On day 3 of conservation, sperm were analyzed and used for AI (n = 174). High sperm quality was observed after storage without a significant difference between the groups (p > 0.05). Moreover, no differences were obtained for AI performance (pregnancy and farrowing rates, and litter size; p > 0.05) and offspring health (growth and blood analysis; p > 0.05). Conclusively, the presence of all ejaculate fractions within the seminal doses does not impair the reproductive performance, reporting important economic savings according to the economic model included here.

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain.

Simple SummaryEfficient collection and cryosurvival of semen from threatened wildlife species is key for the success of artificial reproductive technologies (ARTs). The viscous nature of ejaculates often collected from species such as rhinoceros, elephant, hippopotamus and primate, render the majority of spermatozoa collected useless and is therefore wasted. The enzyme papain has been used to reduce the viscosity of camelid semen but has yet to be tested in wildlife species. Therefore, the current study investigated the ability of papain to improve the yield and quality of spermatozoa collected from viscous fractions of the rhinoceros ejaculate during cryopreservation. Papain increased the quantity of useable spermatozoa collected from ejaculates, as well as the motility prior to freezing. It also improved the post-thaw motility, velocity, linearity and straightness of samples compared to spermatozoa frozen from the sperm-rich fraction of the ejaculate. There was no detrimental effect on membrane characteristics or DNA integrity. These results show that treating rhinoceros ejaculates with papain is able to salvage valuable spermatozoa and improve survival post-thaw, ultimately increasing the success of ARTs and creation of biobanks for the maintenance and survival of threatened species.The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

Alkaline phosphatase in seminal plasma of sperm-rich fraction of semen in fertile and subfertile dogs

Twenty dogs presented for breeding soundness evaluation were selected for the presentstudy. Based on previous six month breeding history and semen quality, these animals weregrouped retrospectively into fertile (n=6) and subfertile (n=14) groups. Three semen collectionswere made from each of these dogs, two weeks apart. Semen evaluation parameters like volume,sperm concentration, progressive motility, viability, morphologically normal sperms, total spermoutput and alkaline phosphatase (ALP) level in the sperm-rich fraction were compared betweenthe two groups. The mean values for progressive motility, sperm concentration, total sperm outputand viability were 84.17 ± 1.54 per cent, 418.33 ± 36.64 million/mL, 645.11±98.43 million and84.92 ± 1.56 per cent, respectively, in the fertile group, while the values for these parameters were45.71 ± 3.47 per cent, 112.07 ± 24.36 million/mL, 190.24±32.45 million and 49.54 ± 2.35 percent, respectively in the subfertile group. The values were significantly higher in fertile group whencompared to subfertile group. The mean ALP (IU/L) was recorded as 168121.67 ± 22443.25 and 49605.86 ± 12669.68 IU/L, respectively in fertile and subfertile dogs. On statistical analysis, a highlysignificant difference (p˂0.01) was noticed between the groups. Though the concentration of ALPwas more than 40,000 IU/L (below which is the proposed cut off value for incomplete ejaculation) inall the dogs under the present study, it was significantly higher in fertile dogs, suggesting a possible relation to fertility.

Effects of age, size and season on cryotolerance of dog semen – A retrospective analysis

In a retrospective analysis of 508 ejaculates from 297 dogs, efficiency of semen cryopreservation and effects of age, season and dog size on characteristics of fresh and cryopreserved semen were evaluated. Volume of the sperm-rich ejaculate fraction increased to 10 years of age, decreased in 10 and 11-year old dogs (P < 0.001) but did not differ among seasons. Total sperm count was less in 10 and 11-year-old dogs (P < 0.001). The percentage of progressively motile, membrane-intact and morphologically normal spermatozoa before cryopreservation was least in 10 and 11-year-old dogs (P < 0.001). Cryopreservation resulted in less progressively motile spermatozoa (P < 0.001) with this being most pronounced in 10 and 11-year-old dogs (cryopreservation x age P = 0.004). The cryopreservation-induced decrease in morphologically normal spermatozoa (P < 0.001) was not affected by dog body weight. Number of cryopreserved AI doses differed among age groups (P < 0.001) and was less in 10 and 11-year-old dogs (median 1.5) compared with younger dogs (6–7 years, median 4.3). When ejaculates were grouped by a threshold of ≥ 35% progressively motile spermatozoa after freezing-thawing, 86.5% of all ejaculates were greater the threshold but this percentage decreased to 66% in 10 and 11-year-old dogs (P < 0.001). In conclusion, sperm cryotolerance is consistent for much of a dog´s lifespan but decreases after a certain age. Dog semen can be cryopreserved successfully throughout the year. Post-thaw semen characteristics were not different among cryopreserved ejaculates obtained from dogs differing in body weight.

Semen Viability Properties of Sexually Stimulated Boars on the Fertilization Capacity of Artificially Inseminated Large White Sows

Background: This study aimed to investigate the influences of sexual preparation on the effects of boars’ semen viability on the fertilization capacity of artificially inseminated sows. After all, boar sires more pigs than farrowed by a sow which the boar has been mated to improve reproductive parameters in response to AI. Methods: The semen viability of boars were studied during various sexual stimulations and analyzed during the study period. Sperm rich fractions were collected and separated at every level of sexual stimulation during the morning (08:30) and the afternoon (14:30) hours, respectively. Artificial insemination was performed following three levels of sexual preparations of boars (0 minutes of sexual restraint (MSR), 5 MSR and 10 MSR) before semen collection. Receptive sows were inseminated and evaluated for fertility traits using non-return rate, farrowing rate and litter size. Result: The non-return rate was recorded as a percentage of sows conceived after insemination over the total number of sows inseminated. The farrowing rate was recorded as a percentage of sows that farrows over the number of sows conceived and litter size as several live piglets per sow. Sexual desire was influenced by sexual preparations and significantly influenced the fertility of the artificially inseminated sows. This study is of practical significance to the animal breeder mainly because boars have greater influence than sows on the average litter size and live piglets. The study concludes that the use of at least 5 to 10 minutes of sexual restraint during the afternoon periods prior to semen collection and artificial insemination is found to be a practical method for optimizing sperm viability and fertility of sows in the intensive system.

Aldose Reductase B1 in Pig Seminal Plasma: Identification, Localization in Reproductive Tissues, and Relationship With Quality and Sperm Preservation.

Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.