Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated ...whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index DFI, mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.
Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the ...majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC–MS/MS and shotgun proteomic analysis by 2D–LC–MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction.
This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.
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•Cross species comparison of the seminal plasma proteomes of seven mammalian species•The similarity of seminal proteomes can be related to the phylogeny of the species.•Beta nerve growth factor is the major protein in alpaca and camel seminal plasma.•Bodhesins and zinc alpha glycoprotein are a signature of small ruminant seminal plasma proteomes.•Nucleobindin-1 and RSVP14 proteins are common to the seven species.
Multiple ovulation and embryo transfer (MOET) technologies are integral to genetic improvement programs in the sheep industries. Despite the protocols being well established, previous findings ...regarding the effects of embryo properties on MOET success remain contradictory. The objective of this study was to determine the effects of embryo developmental stage and quality on embryo viability following transfer to recipient ewes. Data including details of 377 embryos collected from 45 Merino donor ewes were obtained from MOET trials conducted on three separate farms on day 6 after laparoscopic artificial insemination (AI). A total of 270 embryos were classified as being of transferrable grade (grade 1: n = 233; grade 2: n = 37). One or two transferrable grade embryos were transferred to each of 256 synchronised recipient ewes and pregnancy diagnosis was performed on day 36 after embryo transfer. Embryos at the hatched blastocyst stage tended to have greater viability in vivo compared to embryos at the late morula stage (59.0 ± 10.6% vs. 36.2 ± 9.7%; P = 0.083). The viability of grade 1 embryos was greater than that of grade 2 embryos (53.6 ± 7.8% vs. 35.9 ± 10.2%; P < 0.05). The results suggest that the success of the MOET trials was influenced by the transfer of embryos at the late morula stage, almost half of which were classified as grade 2 embryos. These findings highlight the importance of following strict embryo quality grading criteria to inform the most economical management of recipient ewes and maximize pregnancy outcomes.
The modern domestic sow exhibits a period of impaired reproductive performance known as seasonal infertility during the late summer and early autumn months. A reduction in farrowing rate due to ...pregnancy loss is the most economically significant manifestation of this phenomenon. Presently, little is known of the aetiology of seasonal pregnancy loss in the pig. Recent findings represent a major advancement in the understanding of sow reproductive physiology and implicate poor oocyte developmental competence as a contributing factor to pregnancy loss during the seasonal infertility period. It has also been demonstrated that ovarian activity is depressed during the seasonal infertility period. The reduction in oocyte quality is associated with decreased levels of progesterone in follicular fluid during final oocyte maturation in vivo. The recent identification of sow-specific risk factors, such as parity for late pregnancy loss, should improve breeding herd efficiency by allowing producers to tailor management interventions and/or culling protocols that target animals identified as having a greater risk of late pregnancy loss during the seasonal infertility period.
The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from ...slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs. 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs. 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes
The modern domestic sow exhibits a period of impaired reproductive performance during the late summer and early autumn months, known as 'seasonal infertility'. A reduction in farrowing rate due to ...pregnancy loss is the most economically important manifestation of seasonal infertility. The aim of the present study was to determine whether there are changes in oocyte developmental competence associated with season. Ovaries were collected in pairs from sows sourced from commercial piggeries and slaughtered 4 days after weaning during winter and summer-autumn. Following oocyte IVM and parthenogenetic activation, the ability of oocytes from large follicles to form blastocysts was greater in winter (54.94±6.11%) than in summer (21.09±5.59%). During winter, the proportion of oocytes developing to the blastocyst stage from large follicles was significantly higher (54.94±6.11%) than those oocytes from small follicles (23.17±6.02%). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. There was no effect of follicle size on blastocyst formation from those oocytes recovered during summer. Blastocysts derived from small follicles during summer had the lowest number of cells (24.25±1.48) compared with blastocysts derived from large follicles during winter (37.5±1.3; P<0.05). The mean progesterone concentration in follicular fluid collected from small follicles was greater in winter than summer (1235.55±164.47 v. 701.3±115.5nmolL⁻¹, respectively; P<0.001). The mean progesterone concentration in the follicular fluid of large follicles was also greater in winter than in summer (2470.9±169.1 v. 1469.2±156.5nmolL⁻¹, respectively; P<0.001). Regression analysis revealed a positive correlation between progesterone concentration and oocyte developmental competence. The results indicate that porcine oocytes fail to reach their full developmental potential during the period of seasonal infertility, suggesting that the pregnancy losses observed at this time of year may be due to reduced oocyte developmental competence.
Cryopreserved, sex-sorted stallion sperm has been shown to have poor fertility. During this study, the effects of cryoprotectant (glycerol GLY and dimethyl formamide DMF), cryoprotectant ...equilibration time (0, 30, 60, 90, or 120 minutes), and cryoprotectant concentration (2%, 3%, or 4% vol/vol) on stored sex-sorted and stored nonsorted stallion sperm were evaluated. Total motility, viability, and DNA integrity (determined using sperm chromatin structure assay) of sperm were assessed after thawing. Equilibration for 90 minutes improved total motility (33.8%) compared with 0 (28.5%) or 120 minutes (29.8%; P < 0.05), though viability was higher after 120 minutes (33.1%) compared with 0 (30.5%) or 30 minutes (31.0%; P < 0.01). The viability of nonsorted sperm decreased as cryoprotectant concentration increased (P < 0.001), and total motility of nonsorted sperm was higher when DMF alone was used (15.8%, 16.6%, and 24.0% for GLY, GLY and DMF, and DMF respectively; P < 0.001). Sex sorting was detrimental to the postthaw quality of sperm; at 45 minutes after thawing, total motility of nonsorted sperm was higher than that of sex-sorted sperm (37.4% vs. 5.6%; P < 0.001), the viability of sex-sorted sperm was lower than that of nonsorted sperm (12.4% vs. 30.0%; P < 0.001, averaged over postthaw time), and sex-sorted sperm had higher detectable DNA fragmentation index (DFI) (63.6% vs. 11.3%, P < 0.001) and mean DFI (285.1 vs. 211.3, P < 0.001) than nonsorted sperm. The viability of sex-sorted sperm was improved by GLY and DMF or DMF compared with GLY (22.6%, 25.3%, and 19.3%, respectively; P < 0.05), and the DNA integrity of sex-sorted sperm was improved by the use of DMF compared with GLY (detectable DFI, 60.2 vs. 66.8, P < 0.05; and mean DFI, 280.9 vs. 289.2, P < 0.05, respectively). In conclusion, postthaw characteristics of stored sex-sorted and stored nonsorted stallion sperm were improved by the use of DMF as a cryoprotectant, though the parameters to benefit differed between sorted and nonsorted sperm.
Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the role of lipid metabolism and the ...interaction between carbohydrate and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-3 (PZM-3), which contains pyruvate and lactate, 3mM L-carnitine was the only dose that improved cleavage rates compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12mM increased cleavage rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0-3: pyruvate and lactate; Days 4-7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing PZM-3 with 3mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3mM L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo's requirements for carbohydrates are not adequately met.
Niacin deficiency has recently been associated with congenital malformations in humans and embryonic death and resorption in mice (Shi et al. New England Journal of Medicine. 2017; 377(6):544-552). ...Mares suffer from high rates of early embryonic loss (Newcombe. Equine Veterinary Education. 12(2):88-101), the aetiology of which remains unknown. Increasing the levels of nicotinamide adenine dinucleotide (NAD+) through supplementing NAD+ precursors has resulted in an improvement in oocyte quality in mice (Bertoldo et al. Cell Rep. 2020; 30: 1670-1681). However, little is known regarding the requirement of niacin and NAD+ in reproductive processes in the mare. The aim of this study was to determine the effect of oral nicotinic acid (NA) supplements, a form of niacin, on the composition of nicotinamide adenine dinucleotide (NAD+) metabolites in the blood and follicular fluid of mares. Vehicle alone or NA (3 g per os) was administered to 7 mares over a minimum of 3 consecutive days during the oestrous cycle. Blood samples were collected immediately prior tosupplemental feeding and at follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was greater than that in the corresponding plasma (15.0 ± 6.5 vs. 3.3 ± 0.9 ng/µL; p < 0.05) and in the follicular fluid of vehicle-fed mares (1.5 ± 0.5 ng/µL; p < 0.001). Meanwhile, the concentration of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA supplemented mares (1043.9 ± 953.5, 1641.7 ± 256.1 and 5.5 ± 5.1 ng/µL) than in the corresponding plasma (70.8 ± 15.4, 875.6 ± 361.1 and 0.6 ± 0.2, respectively; p < 0.1). The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.
Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI ...of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.
