Acquisition of fertilization competence in guinea pig spermatozoa under different capacitation protocols.

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs.

Female reproduction and the microbiota in mammals: Where are we?

While it is generally accepted that the mammalian vagina contains a site-specific microbiota that plays relevant roles in genital and reproductive health, the existence of an extra-vaginal microbiota in the female reproductive tract (i.e. follicular fluid, oviduct, endometrium, and placenta) is, at least, a matter of controversy. Many conclusions in this field have failed to consider the technical limitations, biases, and confounding factors inherent to next-generation sequencing (NGS) approaches. While this creates uncertainty in the field, there is no doubt this subject is set to be the focus of new research efforts because of its scientific and practical connotations in female reproductive health. The current art state, its limitations, and gaps in our knowledge about the female reproductive tract's microbiota and, particularly, about the microbes of the extra-vaginal environment are presented in this review. Also are discussed possible relationships between the gut and oral microbiota and reproductive events.

Influence of Different Regimes of Moderate Maternal Feed Restriction during Pregnancy of Primiparous Rabbit Does on Long-Term Metabolic Energy Homeostasis, Productive Performance and Welfare.

In this study, a maternal feed restriction (MFR; 105 g/d) in primiparous rabbit does was applied from day 0 to 7 post artificial insemination (AI) (R07, n = 96), from day 7 to 21 post AI (R721, n = 92), from day 0 to 21 post AI (R021, n = 94) or fed ad libitum during whole pregnancy (Control, n= 92). Feed intake (FI) was measured after MFR was over. On day 28 of gestation, fetoplacental development was evaluated (n = 11/group) and the productive parameters of the remaining dams were analyzed. Plasma free tri-iodothyronine (T3) and thyroxine, glucose, insulin, non-esterified fatty acids (NEFA), and corticosterone were analyzed during gestation and lactation (n = 5/group). After MFR, all groups significantly increased their voluntary FI. The longer MFR was, the lower the weight and length of the fetuses, but no long-term effects over litter performance were observed. R021 groups had the lowest T3 and the highest NEFA concentrations during pregnancy and showed insulin resistance at the end of gestation, but during lactation, energy homeostasis was balanced in all groups. MFR did not affect corticosterone concentrations. In conclusion, the ration setting applied slightly involved the energy homeostasis and metabolism of the animals, but their overall metabolic condition, productive performance and welfare were not compromised.

Gestation Food Restriction and Refeeding Compensate Maternal Energy Status and Alleviate Metabolic Consequences in Juvenile Offspring in a Rabbit Model.

Nutritional status during gestation can influence mother and offspring metabolism. Undernutrition in pregnancy affects women in both western and developing countries, and it is associated with a high prevalence of chronic diseases in later life. The present work was conducted in the rabbit model, as a longitudinal study, to examine the effect of food restriction during early and mid-gestation, and re-feeding ad libitum until the end of pregnancy on metabolic status and body reserves of mother and, its association with development and metabolism of fetuses and female offspring to the juvenile stage. Little changes in live body weight (LBW), compensatory feed intake, similar body reserves, and metabolism were observed in dams. Placenta biometry and efficiency were slightly affected, but fetal BW and phenotype were not modified. However, hyperinsulinemia, insulin resistance, and hypertriglyceridemia were demonstrated in pre-term fetuses. In the juvenile period, these changes were not evidenced, and a similar pattern of growth and serum metabolic parameters in offspring of food-restricted mothers were found, except in serum aminotransferases levels, which increased. These were associated with higher liver fibrosis. Maternal food restriction in the early and mid-pregnancy followed by re-feeding in our rabbit model established a compensatory energy status in dams and alleviated potential long-term consequences in growth and metabolism in the offspring, even if fetal metabolism was altered.

Physiological effects on rabbit sperm and reproductive response to recombinant rabbit beta nerve growth factor administered by intravaginal route in rabbit does.

Beta nerve growth factor (ß-NGF) is present in the seminal plasma of some species, including rabbits, acting as an ovulation-inducing factor in camelids. Traditionally, GnRH analogues are used to induce ovulation by intramuscular route when artificial insemination (AI) is performed in rabbit does. A specific rabbit recombinant ß-NGF (rrß-NGF) produced in our laboratory was tested as an alternative method to conventional treatment with GnRH analogues to induce ovulation. In the present work, different concentrations (0, 20, 100 ng/mL and 1, 20 and 100 µg/mL) of rrß-NGF were added to diluted semen to assess its effect on sperm traits (viability and motility parameters). rrß-NG was used also, incorporated to the AI dose, to evaluate ovulation response (LH and progesterone plasma concentrations, ovulation rate (OR) and embryo implantation at Day 7) after intravaginal administration. A negative control group stimulated with an empty catheter, and a positive control group inseminated and intramuscularly treated as usual with GnRH were also set up. Results showed that seminal quality was influenced by rrß-NGF depending on the concentration added, being the highest concentrations tested deleterious for semen. Whereas the highest OR was found in the positive control group (100%), concentrations of 20 ng/mL, 1 µg/mL and 20 µg/mL of rrß-NGF triggered intermediate OR (30, 60 and 42.9%, respectively), and 100 ng/mL and 100 µg/mL had the lowest OR (20 and 14.3%, respectively). Although LH peak was not observed in the first 2 h after AI in the ovulated females from rrß-NGF groups, plasma progesterone significantly increased at Day 7, except in those females treated with 20 and 100 µg/mL. Also, 98.4% of ovulated females were pregnant on Day 7. In conclusion, rrß-NGF added to diluted semen affects seminal quality and provokes ovulation, the development of functional CL and conception by intravaginal route in rabbit does, depending on the concentration added.

Correction: Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

[This corrects the article DOI: 10.1371/journal.pone.0219780.].

Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

In some induced-ovulating species, beta nerve growth factor (ß-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant ß-NGF from rabbit tissue (rrß-NGF) to address the following objectives: 1) to compare rabbit ß-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrß-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrß-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rß-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to ß-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrß-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 µg/mL of the neuropeptide. Addition of this same concentration of rrß-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrß-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.

Characterization of ß-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between ß-NGF and testosterone levels with seminal quality during sexual maturation.

ß-Nerve Growth Factor (ß-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of ß-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. ß-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine ß-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate ß-NGF concentration, and testosterone levels were also assessed in BP. The highest ß-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual ß-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of ß-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, ß-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. ß-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and ß-NGF.

Gene expression and immunolocalization of low-affinity neurotrophin receptor (p75) in rabbit male reproductive tract during sexual maturation.

This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits.

Features of follicle-stimulating hormone-stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles.

OBJECTIVE: To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. DESIGN: Observational, model study. SETTING: Public research unit. ANIMAL(S): Fifteen adult Manchega ewes. INTERVENTION(S): Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. MAIN OUTCOME MEASURE(S): Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. RESULT(S): These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. CONCLUSION(S): Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Multiple factors affecting the efficiency of multiple ovulation and embryo transfer in sheep and goats.

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Ovarian response in sheep superovulated after pretreatment with growth hormone and GnRH antagonists is weakened by failures in oocyte maturation.

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.