Fertility Of Semen Research Articles

Effects of astaxanthin supplementation on the freezability, lipid peroxidation, antioxidant enzyme activities and post-thawing fertility of ram semen

The aim of present study was to determine the effect of astaxanthin supplementation of the semen extender on post-thaw semen quality and fertility. Semen samples were collected from four fertile rams by using an artificial vagina, pooled and diluted with a Tris–citric acid–fructose–yolk extender, supplemented with AST at 0.5, 1, 2, or 4 μM concentraitons. For the control group, semen was processed in the basic extender without AST. Extended semen was aspirated into straws which were cooled to 5 °C and frozen on liquid nitrogen vapor. After 24 h, the straws were thawed in a water bath for 30 s. The results showed that supplementation of the extender with 2 and 4 μM AST increased the sperm viability and plasma membrane integrity in frozen-thawed semen compared to other groups (P < 0.05), whilst there was no significant difference between extenders containing 0.5 and 1 μM AST. Freezing the sperm in the extenders containing 2 and 4 μM AST led to lower percentages of acrosome abnormalities (27.20% ± 0.54 and 28.15% ±0.54, respectively) and malondialdehyde formation (1.70 ± 0.1 and 1.81 ± 0.2 nmol/ml, respectively) in comparison to other treatments (P < 0.05). There was no effect of AST on SOD and GSH-PX activities (P > 0.05). Higher fertility rates were obtained when semen extender contained 2 μM (48.33%) and 4 μM (45.76%) AST compared with control group (21.15%). The results showed that, inclusion of AST in frozen ram semen extenders could improve seminal quality and fertility rate.

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.

Study the Morphometry of Fresh and Cryopreserved Murrah Bull Spermatozoa

Artificial insemination with frozen – thawed spermatozoa was introduced in most of the developing countries more than three decades ago, yet it has not been successfully applied in large scale (Anzar et al., 2003). More than 50% spermatozoa are usually injured by the cryopreservation process (Watson, 1995) injuries due to cryopreservation and more likely due to the increased solute concentration and the formation of intracellular and extracellular ice crystals during cryopreservation (Mazur, 1984) leading to a significant decline in semen quality and alterations in sperm morphometrix. Therefore, the present study was designed to study cryopreservation of buffalo semen and the effect of semen cryopreservation on determinant like semen fertility viz. sperm morphometry. Forty-eight semen samples collected from eight Murrah buffalo bulls maintained at Central semen station, Bhopal were included for the study of physic-morphological characters of neat and cryopreserved semen. Various measurements of the sperm head (length, width, base, ellipticity, elongation, head shape and area) and sperm tail (length of Midpiece and length of tail) were recorded in neat and cryopreserved semen. The average sperm measurements recorded were 7.24 ± 0.06, 4.26 ± 0.05, 2.65 ± 0.79, 11.92 ± 1.53, 43.12 ± 2.79, 61.97 ± 0.50, 25 ± 0.07, 19.13 ± 2.58, 0.59 ± 0.01, and 59.07 ± 0.94, respectively. There was a significant (P<0.05) decline of sperm head length, head width, midpiece length, tail length, total length and head area during the process of cryopreservation. However, no significant difference between width at base, ellipticity, head shape and elongation of the spermatozoa in neat and cryopreserved semen was recorded.

Supplementation of soybean lecithin-based cryopreservation medium with glutathione: Fertility and flow cytometry study of ram frozen-thawed semen

Abstract This study was aimed to evaluate the beneficial effects of reduced glutathione antioxidant in soybean lecithin-based cryopreservation medium on fertility and quality parameters of ram semen. In experiment 1, semen samples were collected from five rams and diluted in the freezing extender contained various concentrations of glutathione as follows: extender without glutathione (G0), extender containing 0.5 mM (G0.5), 1 mM (G1), 2 mM (G2), 4 mM (G4) and 8 mM (G8) glutathione. Motility, membrane functionality, morphology, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation and reactive oxygen species (ROS) concentration were assessed after thawing. In experiment 2, pregnancy, parturition, lambing and twining rates were evaluated after artificial insemination with frozen-thawed semen. In results, G4 showed higher (P ≤ 0.05) total and progressive motility, membrane functionality, acrosome integrity, and lower (P ≤ 0.05) lipid peroxidation and ROS concentration compared to the other groups. Mitochondrial activity and viability were higher (P ≤ 0.05) and late apoptotic-like changes were lower (P ≤ 0.05) in G2 and G4 compared to the other groups. DNA fragmentation and late apoptotic-like changes were higher (P ≤ 0.05) in G8 compared to the other groups. No significant difference was observed among groups in cases of morphology and reproductive performance. In conclusion, supplementation of cryopreservation medium with 4 mM reduced glutathione improves the quality parameters of ram spermatozoa but it was not reflected to the reproductive performance.

Effect of statin on semen quality characteristics.

Statins are lipid-lowering medications widely used to reduce the risk of cardiovascular diseases. Biochemically, they act by decreasing synthesis of cholesterol via inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Since 1992, various research studies have investigated the effect of statins on semen quality characteristics; however, to date, there is no collective summary to such effect. Here, we have systematically discussed and abridged all research studies published in Scopus, PubMed and Web of Science databases that are directly linking statin to semen fertility characteristics using the keywords "statin" versus "sperm" and "semen". In summary, considering the animal studies, statins, in general, were found to ameliorate semen quality characteristics in reproductive detrimental conditions, while, in human males or in in vivo systems with normal reproductive conditions, in general, statins showed negative to blunt effects against semen quality characteristics, mainly sperm motility. However, further research studies, in particular human studies, in this specific research setting is still needed to approve these effects.

A comprehensive overview of bull sperm-borne small non-coding RNAs and their diversity across breeds

BackgroundMature sperm carry thousands of RNAs, including mRNAs, lncRNAs, tRNAs, rRNAs and sncRNAs, though their functional significance is still a matter of debate. Growing evidence suggests that sperm RNAs, especially sncRNAs, are selectively retained during spermiogenesis or specifically transferred during epididymis maturation, and are thus delivered to the oocyte at fertilization, providing resources for embryo development. However , a deep characterization of the sncRNA content of bull sperm and its expression profile across breeds is currently lacking. To fill this gap, we optimized a guanidinium–Trizol total RNA extraction protocol to prepare high-quality RNA from frozen bull sperm collected from 40 representative bulls from six breeds. Deep sequencing was performed (40 M single 50-bp reads per sample) to establish a comprehensive repertoire of cattle sperm sncRNA.ResultsOur study showed that it comprises mostly piRNAs (26%), rRNA fragments (25%), miRNAs (20%) and tRNA fragments (tsRNA, 14%). We identified 5p-halves as the predominant tsRNA subgroup in bull sperm, originating mostly from Gly and Glu isoacceptors. Our study also increased by ~ 50% the sperm repertoire of known miRNAs and identified 2022 predicted miRNAs. About 20% of sperm miRNAs were located within genomic clusters, expanding the list of known polycistronic pri-miRNA clusters and defining several networks of co-expressed miRNAs. Strikingly, our study highlighted the great diversity of isomiRs, resulting mainly from deletions and non-templated additions (A and U) at the 3p end. Substitutions within miRNA sequence accounted for 40% of isomiRs, with G>A, U>C and C>U substitutions being the most frequent variations. In addition, many sncRNAs were found to be differentially expressed across breeds.ConclusionsOur study provides a comprehensive overview of cattle sperm sncRNA, and these findings will pave the way for future work on the role of sncRNAs in embryo development and their relevance as biomarkers of semen fertility.

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = −0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.

Recent advances in preservation of boar semen in liquid state: An overview

Current century of artificial insemination (AI) of female pigs, knowledge about the preservation of boar semen is still very limited. In pig liquid semen preservation was mainly done at 15-20 ˚C for 5 days. The short-term and long-term extenders are used according to the time required for AI. In short-term extender both viability and fertility of semen was restored whether in long-term storage although the viability of semen was maintain but the fertility was extremely reduced with the time of preservation. The present study would try to give an overview about the preservation technique of boar semen in terms of extender, preservation time, preservation temperature, viability and fertility.

The current state of the problem of in vitro gene pool preservation in poultry.

This review presents the current progress in and approaches to in vitro conservation of reproductive cells of animals, including birds, such as cryopreservation and freeze-drying, as well as epigenetic conditions for restoring viable spermatozoa and female gametes after conservation. Cryopreservation is an effective way to preserve reproductive cells of various species of animals and birds. In vitro gene pool conservation is aimed primarily to the restoration of extinct breeds and populations and to the support of genetic diversity in populations prone to genetic drift. It is the combination of ex situ in vivo and ex situ in vitro methods that can form the basic principles of the strategy of animal genetic diversity preservation. Also, use of cryopreserved semen allows faster breeding in industrial poultry farming. Despite numerous advances in semen cryobiology, new methods that can more efficiently restore semen fertility after cryopreservation are being sought. The mechanisms underlying the effect of cryopreservation on the semen parameters of cocks are insufficiently understood. The review reflects the results of recent research in the field of cryopreservation of female and male germ cells, embryonic cells, the search for new ways in the field of genetic diversity in vitro (the development of new cryoprotective media and new conservation technologies: freeze-drying). Molecular aspects of cryopreservation and the mechanisms of cryopreservation influence on the epigenetic state of cells are highlighted. Data on the results of studies in the field of male reproductive cell lyophilization are presented. The freeze-drying of reproductive cells, as a technology for cheaper access to the genetic material of wild and domestic animals, compared to cryopreservation, attracts the attention of scientists in Japan, Israel, Egypt, Spain, and France. There is growing interest in the use of lyophilized semen in genetic engineering technologies. Methods of freeze-drying are developed taking into account the species of birds. Organizational and legal ways of solving the problems of in vitro conservation of genetic resources of farm animals, including birds, are proposed.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos.

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Infertile Semen Bacterial Isolates and their Local Multiple Antibacterial Resistance Pattern in Lagos, Nigeria

Introduction: Infertility affects about 15% of couples globally and male contributory factor accounts for 20-30% of such cases. Antibacterial treatment is recommended for infertility of bacterial aetiology. However, Multidrug Resistance (MDR) of the organisms could impair the effectiveness of such infertility treatment. Aim: The present study researched on the MDR pattern of bacteria from semen of infertile men in Lagos, Nigeria. Materials and Methods: This was a controlled cross-sectional study, where in-vitro antibiotic sensitivity tests were conducted on consecutive bacterial isolates from prospective infertile and fertile semen control groups, using Clinical and Laboratory Standard Institute’s (CLSI) interpretative criteria. Antibacterial resistance was expressed in % and semen quality of “before and after” treatment was compered using t-test, p-value &lt;0.05, at 95% confidence interval. Results: A total of 174 (117 Gram positive and 55 Gram negative and 2 yeast-like cells) mainly Staphylococcus spp. and Escherichia coli were studied, between 2009 and 2014. The Gram-positive cocci showed low resistance to Cefoxitin (9.2%) and Fluoroquinolone (45.9%); while Penicillin showed the highest resistance (98.37%). Within organisms’ total resistance rates of 98.3% Amoxicillin (AMX), 89.7% Cloxacillin (CXC), 87.2% Nalidixic acid (NAL) and 83.7% Sulfonamide (Cotrimoxazole) (COT) were recorded. The most prevalent Gram negative isolate (E.coli), showed 100% resistance to AMX, 95.8% Erythromycin, 95.8% Streptomycin, 91.7% amoxicillin/clavulanic acid, 83.3% tetracycline and 83.3% NAL. The most prevalent Gram positive isolate (S. aureus) had resistance rates 97% for AMX, 92.6% NAL, and 91.2% CXC among others. All MDR strains had MICs ranging 4-256 mg/L to the panel of antibiotics tested. Conclusion: There is concurrence in the pattern of MDR in this study to those seen across the world; however, increasing rates were apparent, probably due to lack of effective control policies. There is need for drug surveillance and control in Nigeria.

Effect of carboxylated poly l-Lysine as a cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull semen

Buffalo bull sperm are more prone to cryo-injuries. Glycerol being the most common permeable cryoprotectant exerts cytotoxic effects on sperm which cause a reduction in fertility. Thus, the exploration of new cryoprotectant is needed. For this purpose, we investigated the effect of carboxylated poly l-Lysine (CPLL) as cryoprotectant used with different concentrations of glycerol on post-thaw sperm motility, kinematics, plasma membrane integrity, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), catalase concentration and in vivo fertility of Nili Ravi buffalo bull semen. In experiment 1, semen samples (n = 15, bulls = 3) were diluted with Tris-citrate-egg yolk extender containing different concentration of CPLL [0% (C0), 0.25% (C0.25), 0.5% (C0.5), 0.75% (C0.75), 1% (C1)]. Each concentration of CPLL was added in extender containing either 7% (G7) or 5% (G5) glycerol. Diluted semen samples were cooled and cryopreserved using standard procedures. Post-thaw total and progressive motility, plasma membrane integrity, acrosome integrity, and MMP were found higher (P < 0.05) in group (G5C0.75) containing 0.75% CPLL and 5% glycerol as compared to the control group (G7C0) and other groups while LPO was recorded lower (P < 0.05) in the same group (G5C0.75). In experiment 2, in vivo fertility was compared between G5C0.75 (5% Glycerol+ 0.75% CPLL; depicted better post-thaw quality) and control group G7C0. Buffaloes were inseminated after 24 h of onset of estrus. Pregnancy diagnosis was performed per rectum at least 60 days post insemination. The fertility rates [56% (58/102) vs. 36% (37/103)] were higher (P < 0.05) in G5C0.75 as compared to the control group G7C0. Based upon these results, this study concludes that the addition of 0.75% CPLL in combination with 5% glycerol in freezing extender improves the post-thaw structure, function and in vivo fertility of Nili Ravi buffalo bull semen.

Evaluation by re-derivation of a paternal line after 18 generations on seminal traits, proteome and fertility

Males from a paternal line selected for growth traits were used to produce semen doses at insemination centres and farms in a breeding scheme for rabbit meat production. The aim of this study was to assess whether a program of selection by daily gain in fattening period changed the seminal traits, plasma and sperm proteome and the fertility of semen when used in artificial insemination. Thirty-nine males from a paternal line were obtained by re-derivation from vitrified embryos with a difference of 18 generations (G21V and G39V). Sperm production parameters, morphological traits, sperm motility parameters and viability were evaluated from ejaculates. Seminal plasma and sperm proteome of three pool ejaculates from 10 mature males of each group were analysed and semen doses were used to inseminate 311 females. Only the percentage of abnormal sperm showed significant differences, with G21V presenting fewer abnormal sperm than G39V (10.5 ± 2.63 vs 23.8 ± 1.98). The discriminant analysis (DA-PLS) showed a clear effect of the generation for plasma and sperm proteome. In seminal plasma, 643 proteins were reported and 64 proteins were differentially expressed, of which 56 were overexpressed in G39V (87.5%). Sperm proteome reported 1360 proteins with 132 differentially abundant proteins. Of the total, 89 proteins were overexpressed in G39V (67.4%). From the 64 and 132 differentially abundant proteins of plasma and sperm, 19 and 26 had a FC>1.5, 12 and 13 of them belonging to the Oryctolagus cuniculus taxonomy, respectively. Despite observing differences in important proteins related to capacitation, sperm motility or immunoprotection and consequently to the fertilization process (TMPRSS2, Serpin family, Fam71f1, ATPase H+ transporting accessory protein 2, carbonic anhydrase 2, UDP-glucose glycoprotein glucosyltransferase 2), no differences in fertility and prolificacy were detected when commercial seminal doses were used for insemination from both male groups. However, overabundance of KIAA1324 protein can be related to the increase in abnormal sperm after selection by growth rate.

113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1×107 spermmL−1. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of &amp;gt;25μm s−1; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with invitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value&amp;lt;0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5mM, vs. 0% for Control); 1 and 5mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1mM βMe: 17.6% vs. 13.8%; for 1.0mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.

111 Fertilizing ability of frozen and freeze-dried semen following intracytoplasmic sperm injection of invitro-matured sheep oocytes

Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P&amp;lt;0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production. Table 1.Pronuclear formation at 17-21h post-injection1 Treatment n FPN MPN 2PN 3PN NPN FT 71 9.66±4.12 4.26±1.48 48.13±2.79a 5.97±4.16 31.98±6.75a FD 65 6.16±2.26 1.39±1.39 20.15±4.14b 10.57±6.59 61.73±6.89b a,bValues in the same column with different superscript letters differ significantly (P&amp;lt;0.05). 1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei. Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.

118 Semen quality and fertilization ability of myostatin-knockout boars

Myostatin-knockout (MSTN−/−) pigs may provide a source of healthy lean protein for human consumption. However, little is known about the effect of this knockout on semen quality, which is important if these pigs are used for production. The purpose of this study is to evaluate the semen quality and fertility of MSTN−/− boars. We generated MSTN−/− boars from Duroc-Landrace-Yorkshire hybrid pig cell lines by somatic cell nuclear transfer, and all 12 boars showed sexual maturation with an obvious “double muscling” phenotype. Semen was collected randomly from three MSTN−/− boars using the gloved-hand technique by one technician and then tested by computer-assisted semen analysis. Semen acrosomal integrity and deformity were measured using Coomassie blue- and eosin-stained smears, respectively. Sperm plasma membrane integrity and mitochondrial activity were evaluated by Hoechst 33342, propidium iodide, and JC-1 multiple staining. The reproductive performance of MSTN−/− boars was evaluated by IVF and by AI. All data were analysed by Student's t-tests. The results showed that the semen color, odor, and pH had no abnormalities. The concentration, motility, plasma membrane integrity, deformity, acrosome integrity, and mitochondrial activity of the semen presented no significant differences from those of the control semen (Duroc). The ejaculation volume of the MSTN−/− boars was significantly lower than that of the control (168.78±6.70 and 223.11±21.21mL, respectively), although the total sperm number was not significantly different. The rate of cleavage and blastocyst formation (247 to 254 oocytes per boar) was not significantly different from those of the control (69.1±0.7 vs. 65.2±1.6%, and 20.2±1.2 vs. 22.8±1.4%, respectively). Seventeen healthy offspring were successfully produced from three sows through AI using semen from one MSTN−/− boar. However, the genotype of piglets has not been tested at present. Thus, MSTN−/− boar may be used as sires, and these pigs are expected to be developed to provide new super-lean meat varieties in the future.

Myoinositol Supplementation of Freezing Medium Improves the Quality-Related Parameters of Dog Sperm.

Simple SummaryThe generation of free radical reactive oxygen species during freeze–thaw procedures is one of the major factors affecting the function and survival of sperm. Myoinositol is the most important natural form of inositol produced in the human body. Researchers have attempted to exploit the antioxidant nature of myoinositol to treat human infertility issues via the improvement of sperm quality traits and fertilization rates. We investigated the potential role of myoinositol neutralizing free radicals produced during the cryopreservation of dog semen. Myoinositol supplementation in the freezing medium resulted in improved quality-related parameters of dog semen including percentage motility, viability, plasma membrane integrity, and chromatin integrity. Improvement in post-thaw semen quality was confirmed by the expression of genes related to apoptosis, nuclear integrity, and reactive oxygen species generation. Oxidative stress during freeze–thaw procedures results in reduced semen fertility. A decrease in free radical levels can improve the post-thaw sperm quality. We examined the effects of myoinositol supplementation in freezing medium on the structure and function of cryopreserved dog sperm. Pooled ejaculates were diluted with buffer without or with myoinositol (1 or 2 mg/mL). Analysis of fresh semen revealed that the optimal concentration of myoinositol was 1 mg/mL, and this concentration was used in further experiments. Post-thaw semen quality in the myoinositol-supplemented group was superior (p < 0.05) compared with that in the control group in terms of motility (57.9 ± 0.4% vs. 47.8 ± 0.2%), sperm viability (57.5 ± 0.5% vs. 44.6 ± 0.6%), intact plasma membrane (56.6 ± 0.4% vs. 46.2 ± 0.6%), and acrosome membrane (59.3 ± 0.5% vs. 51.8 ± 0.5%). In addition, sperm in the myoinositol-supplemented group showed a significantly lower expression of pro-apoptotic (BAX) and mitochondrial reactive oxygen species (ROS) modulator (ROMO1) genes but higher expression of anti-apoptotic (BCL2), and protamine-related (PRM2 and PRM3) genes compared with that in the control group. Therefore, myoinositol supplementation before freezing can protect against oxidative stress and improve post-thaw dog sperm quality.

Semen Quality, Fertility and Testicular Histology of Rabbit Bucks Orally Administrated with Ethanolic Grape Seed Extract

Semen Quality, Fertility and Testicular Histology of Rabbit Bucks Orally Administrated with Ethanolic Grape Seed Extract

Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility

BackgroundThe application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.ResultsOur strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.ConclusionsThese results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Artificial insemination with fresh, liquid stored and frozen thawed semen in dromedary camels.

This study was conducted to evaluate various factors affecting fertility following insemination of dromedary camels. In experiment 1, camels were either bred by natural mating (NM) or inseminated in the body of uterus with whole, split (50:50) or 1 mL of undiluted ejaculate. In experiment 2, camels were inseminated with fresh diluted semen either in the body of the uterus or tip of the uterine horn and at either the time of ovulation induction (0 h), 24 or 30 h later. In experiment 3, camels were inseminated at the tip of the uterine horn with different doses of fresh diluted semen (75, 150 or 300 x 106 motile spermatozoa) or with 150 x 106 motile spermatozoa diluted with different extenders (Green buffer, Optixcell or Triladyl). In experiment 4, camels were inseminated in the tip of the uterine horn with diluted (Triladyl or Optixcell) liquid-stored semen or diluted (Triladyl) frozen-thawed semen consisting of either 300 or 500 x 106 motile spermatozoa. The pregnancy rate in camels bred by NM was similar to camels inseminated with whole undiluted ejaculates whereas insemination with 1 mL undiluted ejaculate resulted in lower pregnancy compared to whole and split undiluted ejaculates (P < 0.05). Deposition of semen in the uterine body resulted in lower pregnancy rates compared to deposition in the tip of the horn (35.3% versus 72.2%, P < 0.05) but insemination at the time of ovulation induction and 24 h later resulted in higher pregnancy rate to camels inseminated at 30 h after induction (68.4 and 70.0% versus 23.5%; P < 0.05). Artificial insemination with 75 x 106 motile spermatozoa resulted in lower pregnancy rates compared to 150 and 300 x 106 motile spermatozoa doses (40.9% versus 65.2 and 70.0%, respectively) and pregnancy rate was not affected by extenders. Insemination of chilled motile spermatozoa stored in either Triladyl or Optixcell resulted in similar pregnancy rates, regardless of insemination dose, although an upward trend with increasing sperm number was apparent (Triladyl; 11.1% versus 21.1% and Optixcell; 5.9% versus 12.5%, for 300 x 106 and 500 x 106 groups, respectively; P > 0.05). No pregnancies were obtained with frozen thawed semen. In conclusion, this study demonstrated that the success of camel AI is highly dependent on sperm dose, location of semen deposition, timing of insemination and semen type. Further studies are required to determine the reason for the compromised fertility of preserved semen despite apparent high in vitro quality.