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Simple Summary Knowing which bacteria dominate vaginal microbiota and its variation throughout the cycle is important to study how to prevent reproductive diseases. In women, vaginal microbiota is dominated by Lactobacillus but this does not happen in other animals. Little is known about equine vaginal microbiota. The aim of this study was to describe the dynamics of equine vaginal microbiota during the ovarian cycle. Eight healthy adult Arabian mares were used to characterize vaginal microbiota by standard microbiologic and metagenomic procedures. The abundance of Lactobacillus was < 2% by both methods, meaning that equine vaginal microbiota was not dominated by these bacteria. Dominant bacteria included other genera such as Porphyromonas and Campylobacter among others. No changes in vaginal microbiota composition were found, suggesting that equine vaginal microbiota was stable throughout the ovarian cycle. Abstract Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Simple Summary The use of intravaginal devices may generate retention of vaginal secretions and abnormal discharges at device withdrawal, which may create changes in the normal vaginal bacterial microbiota. These changes in vaginal microbiota could allow the growth of opportunistic pathogenic bacteria which can require antibiotic treatment. The addition of antibiotics on sponges poses a risk of milk contamination. In dairy cows, it has been observed that the use of lactic acid bacteria (LAB) confers a health benefit to the reproductive tract against bacterial infections. However, LAB use in intravaginal sponges in sheep has not been reported. The aims of this study were to describe the dynamics of the ewes’s vaginal cultivable microbiota during a classic long-term estrous synchronization protocol and to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. The present results suggest that LAB infusion in the ewe’s vagina do not affect general health status and did not reduce fertility during natural mating. Abstract Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.

Contents The objective of this study was to determine the effect of exogenous progesterone administration at ovulation and during the early development of the CL, on its future sensitivity to a single administration of PGF2a in mares and cows. Horse Retrospective reproductive data from an equine clinic in the UK during three breeding seasons were used. Mares were divided into: control group, cycles with single ovulations; double ovulation group cycles with asynchronous double ovulations; and PRID group: cycles with single ovulations and treatment with intravaginal progesterone device (CIDR) immediately after the ovulation. All mares were treated with d-cloprostenol (PGF) at either: (i) 88 hr; (ii) 96 hr; (iii) 104 hr; or (iv) 112 hr after the last ovulation. Cattle A total of nine non-lactating Holstein cows were used. All cows were administered PGF14 d apart and allocated to one of two groups control group GnRH was administered 56 hr after the second PGF administration. CIDR group CIDR was inserted at the same time of GnRH administration. All cows were administered PGF at 120 hr post-ovulation. The complete luteolysis rate of mares with double ovulation (66.7%) and those treated with exogenous progesterone (68.4%) was significantly higher than the rate of mares with single ovulation (35.6%) at 104 hr. In the cow, however, the treatment with CIDR did not increase the luteolytic response in cows treated at 120 hr post-ovulation. In conclusion, the degree of complete luteolysis can be influenced by increasing the concentration of progesterone during the early luteal development in mares.

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 &mu, g/mL of enrofloxacin or 0.0625 &mu, g/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 &times, 106 colony forming units (CFU)/mL or 3.24 &times, 108 CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p &lt, 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.

Este artículo se encuentra disponible en la siguiente URL: https://www.tandfonline.com/doi/full/10.1080/09712119.2019.1599899 The present study indicates that short-term progestagen-based protocols for synchronization of estrus and ovulation in sheep involving 7 days of progestagen insertion with administration of prostaglandin F2α at either insertion or removal of the progestagen sponge resulted in 80–90% fertility during the breeding season, while a classical long-term protocol of 14 days of progestagen insertion resulted in 77% fertility. During the non-breeding season, fertility was significantly higher for the 7-day protocol with prostaglandin administration at sponge insertion (79.2%) and for the 14-day protocol (80%) than for the 7-day protocol with prostaglandin administration at sponge removal (59.1%; P = 0.018). Prolificacy, in contrast, varied significantly with genotype, being higher in prolific breeds, but it did not vary with progestagen protocol or breeding season. These results suggest that short-term progestagen-based treatments can provide similar reproductive efficiency as long-term treatments, which may help practitioners reduce welfare and health issues while maintaining productivity.

Este artículo se encuentra disponible en la página web de la revista en la siguiente URL: https://onlinelibrary.wiley.com/doi/abs/10.1111/rda.13317 Este es el pre-print del siguiente artículo: Martínez-Ros, P., Astiz, S., García-Roselló, E., Ríos-Abellán, A. & González-Bulnes, A. (2019). Onset of estrus and preovulatory LH surge and ovulatory efficiency in sheep after short-term treatments with progestagen-sponges and progesterone-CIDRs. Reproduction in Domestic Animals, vol. 54, n. 2 (feb.), pp. 408-411, que se ha publicado de forma definitiva en https://doi.org/10.1111/rda.13317 This is the pre-peer reviewed version of the following article: Martínez-Ros, P., Astiz, S., García-Roselló, E., Ríos-Abellán, A. & González-Bulnes, A. (2019). Onset of estrus and preovulatory LH surge and ovulatory efficiency in sheep after short-term treatments with progestagen-sponges and progesterone-CIDRs. Reproduction in Domestic Animals, vol. 54, n. 2 (feb.), pp. 408-411, which has been published in final form at https://doi.org/10.1111/rda.13317 The present study supports that short-term (7-days) protocols based on progestagen-impregnated sponges and progesterone-loaded CIDRs are equally effective to induce ovulatory response in sheep. There were no significant differences in the onset of estrus behavior (32.0±6.0 and 33.8±4.0h after device withdrawal for sponges and CIDRs, respectively; p > .05) and preovulatory LH discharge (5.1 ± 2.1 and 5.8 ± 3.3h after onset of estrus behavior for sponges and CIDRs, respectively; p > .05). These features are similar to previously described for classical long-term (12-14 days) treatments. Hence, short-term CIDR-based protocols may be implemented using the same time-intervals for insemination than sponge-based and long-term protocols. Preprint

Este artículo se encuentra disponible en la página web de la revista en la siguiente URL: https://www.sciencedirect.com/science/article/abs/pii/S0378432018306626?via%3Dihub This is the pre-peer reviewed version of the following article: Martinez-Ros, P., Astiz, S., Garcia-Rosello, E., Rios-Abellan, A. & Gonzalez-Bulnes, A. (2018). Effects of short-term intravaginal progestagens on the onset and features of estrus, preovulatory LH surge and ovulation in sheep. Animal Reproduction Science, vol. 197 (oct.), pp. 317-323, which has been published in final form at https://doi.org/10.1016/j.anireprosci.2018.08.046. Este es el pre-print del siguiente artículo: Martinez-Ros, P., Astiz, S., Garcia-Rosello, E., Rios-Abellan, A. & Gonzalez-Bulnes, A. (2018). Effects of short-term intravaginal progestagens on the onset and features of estrus, preovulatory LH surge and ovulation in sheep. Animal Reproduction Science, vol. 197 (oct.), pp. 317-323, que se ha publicado de forma definitiva en https://doi.org/10.1016/j.anireprosci.2018.08.046. In the present study, there was a comparison among classical long-term progestagen (fluorogestone acetate) protocols for synchronization of estrus and ovulation (14 days; group FGA14, n = 9 ewes) and short-term protocols based on 7 days of progestagen treatment plus a dose of prostaglandin F at either insertion (PG-FGA7, n = 11) or removal (FGA7-PG, n = 12). There were no significant differences in the ovulation rate and progesterone secretion among treatments. The FGA7-PG group, however, had a similar percentage of ewes expressing estrous behavior than the group FGA14 (90.9 and 100%, respectively, with a trend for a lesser percentage in the PG-FGA7 group, 63.6%) and about 90% of the ewes in the FGA7-PG group had the preovulatory surge release of LH 8 h after the onset of estrous behavior. These features may be related to a greater number of preovulatory follicles during growing phases (P < 0.05) and a greater plasma estradiol concentration (P < 0.05) in this group than in the classical 14-day group, which suggest these are more functional preovulatory follicles. In conclusion, therefore, the use of the FGA7-PG treatment may favor efficiency of progestagen-based protocols for reproductive management. Preprint

The 6-d timed artificial insemination protocol has been designed to advance luteolysis after the first administration of GnRH so that the preovulatory follicular diameter at second GnRH is reduced and thereby pregnancy outcome may be improved. To achieve an earlier and complete luteolysis (5 to 6 d after the first GnRH treatment), an extra PGF(2α) treatment must be administered to cows 24 h after the initial PGF(2α) treatment. Although the use of 2 PGF(2α) treatments increases labor costs resulting from the increased handling of cows, no alternative and efficient protocol with a single PGF(2α) treatment has been found to date. The objective of this study was to compare the effect of a modified 6-d synchronization protocol on the luteolytic response and final preovulatory follicle diameter. The study followed a crossover design: 14 nonlactating dairy cows were included in 2 treatment doses. All cows received a presynchronization treatment consisting of 2 administrations of a PGF(2α) analog (PGF) 14 d apart followed by treatment with GnRH 11 d later. After the first GnRH administration, one treatment consisted of 150 µg of d-cloprostenol 5 and 6 d later (split dose) and the other treatment consisted of 375 µg of d-cloprostenol as a single dose 6 d after the first GnRH (single large dose). All cows were then treated with a second GnRH 8 d after the first. The luteolytic response to treatment was evaluated by blood progesterone concentration and CL area regression -1 to 3 d relative to the last PGF treatment obtained by ELISA and ultrasonography, respectively. Fewer cows of the split dose tended to have complete luteolysis 3 d after the last PGF treatment compared with the cows of the single large dose (35.7 and 64.3%, respectively). The final preovulatory diameter of the dominant follicle was similar in cows from the split dose and single large dose (13.7 ± 0.3 and 13.1 ± 0.5mm, respectively). Our results support the modification of the 6-d synchronization protocol by administering a single high dose of PGF 6 d after GnRH (with the subsequent reduction in labor resulting from reduced handling of animals) without detrimental effects on the luteolytic response of dairy cows and preovulatory diameter of the dominant follicle compared with the original protocol. However, this modification of the 6-d synchronization protocol should be tested in a large field study involving fertility data with lactating cows before its use can be recommended.

Oviductal microenvironment generally provides better condi-tions for early embryo development than the conventionalin vitro system. In an attempt to simulate the oviductconditions or the main potentially influencing factors, theeffect was studied of a bovine oviductal fluid (bOF) treatmentapplied prior to IVF on (i) IVF parameters, (ii) cleavage rate,(iii) blastocyst yield and (iv) blastocyst quality. Embryo qualitywas assessed by morphological embryo quality and relativetranscript abundance of several developmental genes in bovineblastocysts. Furthermore, to study the effect of bOF withoutthe male effect and zona–sperm interaction, artificially acti-vated metaphase II oocytes were also treated with bOF.In vitro-matured bovine oocytes from abattoir ovaries weretreated or untreated with bOF for 30 min and then washedprior to IVF or activation. Subsequently, in vitro-fertilized andparthenogenetic embryos were in vitro cultured for 7 to 8 days.The bOF treatment had no effect on fertilization parameters,cleavage, blastocyst rates both on parthenogenetic and IVFbovine embryos and neither on morphological quality of IVFblastocysts. G6PD and SOD2 genes from IVF blastocystsshowed significant changes in their expression after a bOFtreatment. Significant differences were found for the expressionof SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes betweenexcellent or good blastocysts (Grade 1) and fair blastocysts(Grade 2). To our knowledge, this is the first study thatevaluates the effect of bOF oocyte treatment on fertilizationparameters, development and quality of bovine embryos.

A single treatment with PGF2α is assumed to have no luteolytic effect on cows with corpora lutea < 5 days old. The objective of this study was to determine the effect of a single high dose of PGF2α administered to dairy cattle on the morphology and function of the early CL. The study followed a crossover design with a treatment cycle in which 50 mg of dinoprost were administered 3.5 days postovulation and a control untreated cycle. Ultrasound examination and blood samples were performed during the two consecutive cycles. Corpus luteum (CL) diameter, progesterone concentration, and follicular dynamics characteristics were compared between control and treated cycles. Two of nine cows (22%) developed full luteolysis. The remaining seven cows (78%) had partial luteolysis with a decrease (P < 0.05) in progesterone concentration and CL diameter for two and 12 days post-treatment, respectively. The interovulatory interval of treated cycles (19.7 ± 2.4 days) was not different (P > 0.05) from that of controls (23.8 ± 0.9 days). The transient reduction in progesterone of cows with partial luteolysis had no effect on the proportion of cows with two or three follicular waves, follicle growth rate, or preovulatory diameter (P > 0.05). Two cows developed ovarian cystic degeneration during the PGF2α-induced cycle. In conclusion, the treatment of cows with a high dose of PGF2α 3.5 days postovulation induced some degree of luteolysis in all treated cows. This resulted in partial luteolysis in 78% of treated animals and in full luteolysis in the remaining 22%. © 2011 Elsevier Inc.

Here, we describe a 3-month-old XX male French Bulldog. The diagnosis was based on the clinical signs, gonadal histology and cytogenetic analysis. Additionally, the dog was confirmed to be Sry negative by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR). Canine Sry-negative XX sex reversal is a disorder of gonadal development where individuals who have a female karyotype develop testes or ovotestes. To our knowledge, this case is the first XX male sex reversion described in a French Bulldog.

Intracytoplasmic sperm injection (ICSI) is a powerful technique in the field of assisted reproduction (ART) and provides exciting opportunities for studying the basic mechanisms of fertilization and early embryo development. Nevertheless, its application in agriculture and conservation biology has been greatly hampered by the low success rate reported for this method in respect of economically important species. Specifically, the rates of blastocyst formation and live newborn are greatly reduced when zygotes are generated by ICSI. Except for humans, ICSI remains a low efficiency technology in comparison with alternatives such as in vitro fertilization (IVF) and its application is less widespread. In this paper, we discuss the present status, applications and factors affecting ICSI in pigs and other species.

Intracytoplasmic sperm injection (ICSI) in pigs is a technique with potential application in diverse fields of animal production and biomedicine. Even though there are some cases of live offspring resulting from this technique, its yield is still quite low compared to other species. The aim of this study was to evaluate different factors affecting the ICSI performance. This was done by studying (1) the sequence of culture media for the oocytes after injection; (2) modifications in the in vitro maturation system (IVM) through meiotic inhibitors such as roscovitine, and changes in the IVM time; (3) oocyte activation through injection of inositol triphosphate (InsP(3)) together with the sperm. In vitro matured oocytes were employed. All the ICSI experiments were performed with fresh ejaculated semen. Results showed that porcine ICSI zygotes give an improved proportion of two-cell embryos using the sequence IVF medium-embryo culture medium (NCSU-23) rather than transferring directly to NCSU-23. Pronuclear formation ability was not affected by prematuration, but a faster embryo development was observed in roscovitine treated oocytes. In relation to IVM times, oocytes matured for 36 h can achieve better fertilization percentages than oocytes matured for 44 h. These results were independent of the roscovitine treatment. Finally, no influence on embryo development was observed until the blastocyst stage with the use of the InsP(3) as an exogenous activating factor.

In pigs, intracytoplasmic sperm injection (ICSI) efficiency is still poor. The inadequate deconden - sation of the sperm chromatin, its transformation into the male pronucleus (MPN) together with the subsequent inability to activate the oocyte, seem to be the main causes of the low ICSI efficiency. In order to improve the MPN formation we took two different approaches. On the one hand, the in vitro culture (IVC) medium post- ICSI was supplemented with 1.71mM cysteine (CYS). Alternatively, the sperm membrane was digested with Triton X-100 (TX) before ICSI, to improve the exposure of the sperm chromatin to the oocyte cytoplasm. After 6 h post-ICSI, the activation rate was significantly higher in TX group (70.0%) compared with CYS and control groups (42.2% and 48.9%, respectively; P < 0.05). However, no significant differences between the three groups were observed in terms of the number of pronuclei, 2PN (oocytes with 2 pronuclei and no visible sperm), and 1PN + sperm (oocytes with 1 pronucleus and one sperm head). At 22 h post-ICSI, the activation rates were similar in TX, CYS, and control groups (73.1, 78.9, and 75.7%, respectively). In addition, we did not observe significant differences between TX, CYS, and control groups for the number of pronuclei, 2PN (52.6, 56.7, and 50%, respectively) or 1PN + sperm (21.1, 33.3, and 32.1%, respectively). While no cleavage was observed in the CYS group, no significant differences in the cleavage rate were observed between control (21.3%) and TX (10.5%) groups. In summary, and under our conditions, neither CYS supplement, nor sperm TX pre-treatment were able to improve MPN formation at 6 and 22 h post-ICSI. However, the sperm TX pre-treatment improved oocyte activation at 6 h post-ICSI, although 22 h post-ICSI such a beneficial effect did not persist.

Luteolysis is a key event in cattle reproduction. A standard dose of exogenous PGF(2α) will induce full luteolysis in the majority of cows with a matured corpus luteum (CL). However, this will not occur in cows with a CL

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca(2+) concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca(2+) concentration contained in the injection medium. in Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation. (C) 2011 Elsevier Inc. All rights reserved.

Intracytoplasmic sperm injection-sperm-mediated gene transfer (ICSI-SMGT) is a useful tool for the production of transgenic mice but is still rather inefficient in farm animals. In the current study, we evaluated the effect of the sperm treatments on the efficiency for producing enhanced green fluorescent protein (EGFP)-expressing pig embryos by ICSI-SMGT. Four different sperm treatments were assayed (1) fresh (control), (2) frozen-thawing (FT), (3) quick freezing without cryoprotectant agents (QF), and (4) Triton X-100 treatment (TX-100). First, we evaluated the DNA-binding ability and the viability of sperm under the different treatments coincubated with exogenous DNA (EGFP) by flow cytometry. Second, we evaluated the embryo production rate and the efficiency in transgene expression in embryos after using these spermatozoa to fertilize oocytes by ICSI. Sperm treatment significantly increased DNA-binding capacity but reduced sperm viability compared with that of the control group. Treatments damaging the spermatozoa's membranes (QF and TX-100) resulted in a greater capacity of sperm binding exogenous DNA than that after FT treatment (P < 0.01). Similar rates of EGFP-expressing embryos were obtained from the control, FT, and TX-100 groups (37.04 ± 3.52%, 43.54 ± 5.41%, and 29.03 ± 8.29%, respectively), but were significantly higher in the QF group (80.43 ± 5.91%). These results demonstrate that the integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions between an injected exogenous DNA and the sperm chromatin. However, severe sperm treatments such as QF and TX-100 may damage the sperm nucleus, induce DNA fragmentation, and/or lead to chromosomal breakage with a detrimental effect on further embryonic development. © 2009 Elsevier Inc. All rights reserved.