Description of the vaginal microbiota in nulliparous ewes during natural mating and pregnancy: preliminary signs of the male preputial microbiota modulation.

The vaginal microbiota plays a key role in animals' health. Understanding its diversity and composition and associated changes occurring through the reproductive cycle represents valuable knowledge to disclose the mechanisms leading to dysbiosis and eventually to infection. Even if the human vaginal microbiota has been thoroughly studied, scarce research has been conducted on the vaginal microbiota of livestock. In this study, 16S rRNA gene-based sequencing was performed on vaginal samples of ten nulliparous ewes at three different sampling points: before the estrus synchronization protocol (T0), at the time of estrus before mating (Testrus), and the day of the pregnancy diagnosis (Tpreg). Preputial samples from the three males collected pre and post-mating were also analyzed. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla in vaginal samples. The most abundant genera were Porphyromonas, Anaerococcus, and Peptinophilius. Vaginal microbiota biodiversity decreased during pregnancy. Tenericutes (Ureaplasma spp.) increased significantly at Tpreg in both pregnant and non-pregnant ewes. Differences were observed between pregnant and non-pregnant ewes at Tpreg where pregnant ewes had a significantly higher abundance of Actinobacillus spp. and Ureaplasma spp. Ewes that were diagnosed with pregnancy at Tpreg showed a decreased abundance of gram-negative bacteria such as Bacteroidales, Campylobacterales, and Enterobacteriales. In addition, a significant decrease in the relative abundances of genera within Firmicutes, such as Alloicoccus (Lactobacillales), Atopostipes (Lactobacillales), and an uncultured bacteria W5053 from Family XI (Firmicutes, Clostridiales) was observed in non-pregnant ewes at Tpreg. The four most abundant phyla in the rams' prepuce were the same as in the ewes' vagina. The most abundant genus was Corynebacterium. No major differences were observed in the ram's preputial microbiota between pre and post-mating samples. Nevertheless, the differences in the taxonomic composition of ewes' vaginal microbiota between Testrus and Tpreg could be explained by the exposure to the preputial microbiota. This study offers new insights into the effects of several key steps of the ewe's reproductive cycle such as estrus-synchronization protocol, mating, and pregnancy on ovine vaginal microbiota. The knowledge of the microbiota dynamics during the reproductive cycle can help improve the reproductive outcomes of dams by identifying biomarkers and putative probiotics.

Ruminant-associated Listeria monocytogenes isolates belong preferentially to dairy-associated hypervirulent clones: a longitudinal study in 19 farms.

Studies have shown that ruminants constitute reservoirs of Listeria monocytogenes, but little is known about the epidemiology and genetic diversity of this pathogen within farms. Here we conducted a large-scale longitudinal study to monitor Listeria spp. in 19 dairy farms during three consecutive seasons (N = 3251 samples). L. innocua was the most prevalent species, followed by L. monocytogenes. Listeria monocytogenes was detected in 52.6% of farms and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was most prevalent in environmental samples. Sixty-one different L. monocytogenes cgMLST types were found, 28% common to different animals and/or surfaces within the same farm and 21% previously reported elsewhere in the context of food and human surveillance. Listeria monocytogenes prevalence was not affected by farm hygiene but by season: higher prevalence was observed during winter in cattle, and during winter and spring in sheep farms. Cows in their second lactation had a higher probability of L. monocytogenes faecal shedding. This study highlights dairy farms as a reservoir for hypervirulent L. monocytogenes.

Vaginal Microbiota Is Stable throughout the Estrous Cycle in Arabian Maress.

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.

Vaginal Microbiota Changes During Estrous Cycle in Dairy Heifers.

The vaginal microbiota plays an important role in the health of dairy cattle, and it could be manipulated for the prevention and treatment of reproduction-related infections. The present study profiles and compares the vaginal microbiota of healthy dairy heifers during the estrous cycle focusing the results in follicular (estrus) and luteal (diestrus) phases using 16S rRNA sequencing of the V3-V4 hypervariable region. Twenty 13-16-months-old virgin dairy heifers from a single farm were included in this study. Vaginal swabs and blood samples were obtained during estrus (6-8 h before artificial insemination) and diestrus (14 days after insemination). Estrus was evaluated by an activity monitoring system and confirmed with plasma progesterone immunoassay. Results showed that the taxonomic composition of the vaginal microbiota was different during the follicular and luteal phases. At the phylum level, the most abundant bacterial phyla were Tenericutes, Firmicutes, and Bacteroidetes which comprised more than 75% of the vaginal microbiota composition. The next more abundant phyla, in order of decreasing abundance, were Proteobacteria, Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Patescibacteria. Together with Tenericutes, Firmicutes, and Bacteroidetes represented more than 96% of the bacterial composition. Ureaplasma, Histophilus, f_Corynebacteriaceae, Porphyromonas, Mycoplasma, Ruminococcaceae UCG-005, were the most abundant genera or families. The results also showed that the vaginal microbiota of dairy heifers was non-lactobacillus dominant. The genus Lactobacillus was always found at a low relative abundance during the estrous cycle being more abundant in the follicular than in the luteal phase. Despite more research is needed to explore the potential use of native vaginal microbiota members as probiotics in dairy heifers, this study represents an important step forward. Understanding how the microbiota behaves in healthy heifers will help to identify vaginal dysbiosis related to disease.

The Addition of Lactobacillus spp., Enrofloxacin or Doxycycline Negatively Affects the Viability of Mycoplasma bovis in Diluted Bovine Semen.

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 µg/mL of enrofloxacin or 0.0625 µg/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 × 106 colony forming units (CFU)/mL or 3.24 × 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 < 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.

Use of Probiotics in Intravaginal Sponges in Sheep: A Pilot Study.

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.

Onset of estrus and preovulatory LH surge and ovulatory efficiency in sheep after short-term treatments with progestagen-sponges and progesterone-CIDRs.

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 ± 4.0 hr after device withdrawal for sponges and CIDRs, respectively; p > 0.05) and preovulatory LH discharge (5.1 ± 2.1 and 5.8 ± 3.3 hr after onset of estrus behavior for sponges and CIDRs, respectively; p > 0.05). 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.

Effects of short-term intravaginal progestagens on the onset and features of estrus, preovulatory LH surge and ovulation in sheep.

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 F2α 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.

Analysis of RhoE expression in the testis, epididymis and ductus deferens, and the effects of its deficiency in mice.

Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15-day-old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild-types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild-types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild-types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.

Analysis of different factors influencing the intracytoplasmic sperm injection (ICSI) yield in pigs.

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.

Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs.

The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 muM and 5 muM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non-artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.