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Lactic acid-producing bacteria (LAB) are being widely studied due to their probiotic potential. The aim of the present study was to determine and identify the presence of LAB from canine vaginal samples, as well as to evaluate their probiotic in vitro potential. Ninety-four bitches were included in the study. Vaginal samples were obtained by means of a sterile swab and streaked on Man Rogosa Sharpe agar plates. A total of 100 LAB strains were obtained and submitted to Gram stains and basic biochemical tests, which included catalase, oxidase and haemolysis tests. Thirteen strains belonging to the genera Lactobacillus (n = 10), Lactococcus (n = 2) and Pediococcus (n = 1) were selected as potential probiotics and further subjected to evaluation of resistance to gastrointestinal conditions (pH, lysozyme, bile salts and hydrogen peroxide) and safety and efficacy in vitro (resistance to antibiotics and antimicrobial capacity). Only three strains, one Lactobacillus lactis and two Lactobacillus plantarum, accomplished the requirements for being considered as potential in vitro probiotics.

The interaction between cells and the extracellular medium is of great importance, and drastic changes in extracellular solute concentrations drive water movement across the plasma membrane. Aquaporins (AQPs) are a family of transmembrane channels that allow the transport of water and small solutes across cell membranes. Different members of this family have been identified in gametes. In sperm, they are relevant to osmoadaptation after entering the female reproductive tract, which is crucial for sperm motility activation and capacitation and, thus, for their fertilizing ability. In addition, they are relevant during the cryopreservation process, since some members of this family are also permeable to glycerol, one of the most frequently used cryoprotective agents in livestock. Regarding oocytes, AQPs are very important in their maturation but also during cryopreservation. Further research to define the exact sets of AQPs that are present in oocytes from different species is needed, since the available literature envisages certain AQPs and their roles but does not provide complete information on the whole set of AQPs. This is of considerable importance because, in sperm, specific AQPs are known to compensate the role of non-functional members.

Telomere length has attracted much interest as a topic of study in human reproduction; furthermore, the link between sperm telomere length and fertility outcomes has been investigated in other species. This biomarker, however, has not been much explored in other animals, such as pigs, and whether it is related to sperm quality and fertility outcomes remains unknown. The present work aimed to determine the absolute value of telomere length in pig sperm, as well as its relationship to sperm quality parameters and embryo development. Telomere length was determined through quantitative fluorescence in situ hybridization (qFISH) in 23 pig sperm samples and data were correlated to quality parameters (motility, morphology, and viability) and in vitro fertilization outcomes. We found that the mean telomere length in pig sperm was 22.1 ± 3.6 kb, which is longer than that previously described in humans. Whilst telomere length was not observed to be correlated to sperm quality variables ( p > 0.05), a significant correlation between telomere length and the percentage of morulae 6 days after in vitro fertilization was observed (r s = 0.559; 95% C.I. = (-0.007 to 0.854); p = 0.047). Interestingly, this correlation was not found when percentages of early blastocysts/blastocysts (r s = 0.410; 95% C.I. = (-0.200 to 0.791); p = 0.164) and of hatching/hatched blastocysts (r s = 0.356; 95% C.I. = (- 0.260 to 0.766); p = 0.233) were considered. Through the separation of the samples into two groups by the median value, statistically significant differences between samples with shorter telomeres than the median and samples with longer telomeres than the median were found regarding development to morula (11.5 ± 3.6 vs. 21.8 ± 6.9, respectively) and to early blastocyst/blastocysts (7.6 ± 1.4 vs. 17.9 ± 12.2, respectively) ( p < 0.05). In the light of these results, sperm telomere length may be a useful biomarker for embryo development in pigs, as sperm with longer telomeres lead to higher rates of morulae and blastocysts.

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E 2 (PGE 2 ) (only EXP 1), prostaglandin F 2α (PGF 2α ), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF 2α was lower ( p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15.

Endometritis is one of the main causes of infertility in mares. In the present study, 363 mares with a history of repetitive infertility, and positive endometrial cytology and/or vaginal discharge were included. An endometrial swab for microbiological purposes plus sensitivity test was obtained from each mare. A positive culture was obtained in 89% of mares. The main isolated genera were Staphylococcus (25.1%), Streptococcus (18.2%), Escherichia (17.3%) and Pseudomonas (12.1%). With regard to species, the most isolated microorganism was Escherichia coli (17.3%), Staphylococcus spp. (15.6%) and Streptococcus spp. (13.5%). Sensitivity tests showed that the most efficient antimicrobial was amikacin (57.3% of cultures), followed by cefoxitin (48.6%) and gentamicin (48.3%). When sensitivity test was analyzed in terms of Gram+ and Gram- bacteria, Gram+ were highly resistant to cephaloridine (77.3% of cultures), apramycin (70.8%) and penicillin (62.3%), whereas Gram- were highly resistant to penicillin (85.8%), followed by cephaloridine (78.9%). In conclusion, the present study shows the most prevalent microorganisms isolated from equine endometritis, which were found to be resistant to β-lactam antimicrobials. Likewise, these results highlight the significance of performing microbiological analyses as well as sensitivity tests prior to applying an antimicrobial therapy.

While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3-10, 10-30, 30-50, 50-100, and >100 kDa), affect sperm-PMN binding. Sperm motility, viability, and sperm-PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30-50 kDa or 50-100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30-50 and 50-100 kDa proteins, we suggest that both fractions are involved in the control of the jenny's post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm-PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.

Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored at 4 °C for 24 h. Thereafter, each diluted fraction was split into five equal aliquots and subsequently packed into 0.5-mL straws of red, blue, yellow, white, or transparent color. Straws were either nonirradiated (control) or irradiated with a light-dark-light pattern of 3-3-3 (i.e., light: 3 min, dark: 3 min; light: 3 min) prior to evaluating sperm motility, acrosome and plasma membrane integrity, mitochondrial membrane potential, and intracellular ROS and calcium levels. Our results showed that irradiation increased some motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrities of the plasma membrane and acrosome. Remarkably, the extent of those changes varied with the color of the straw and the extender used; the effects of irradiation were more apparent when sperm were diluted with Equiplus extender and packed into red-colored straws or when samples were diluted with Kenney extender and packed into transparent straws. As the increase in sperm motility and intracellular ROS levels was parallel to that of mitochondrial activity, we suggest that the impact of red light on sperm function relies upon the specific rates of energy provided to the mitochondria, which, in turn, vary with the color of the straw and the turbidity/composition of the extender.

Acute phase proteins (APP) are biomarkers of systemic inflammation, which allow monitoring the evolution of diseases, the response to treatments, and post-operative complications. Ovariectomy (OVE) is frequently performed in veterinary medicine and can be a useful model to evaluate surgical trauma and inflammation in the bitch. The objective was to investigate and compare the acute phase response (APR) after applying three different OVE techniques by measuring serum levels of C-reactive protein (CRP), haptoglobin (Hp), albumin (Alb), and paraoxonase-1 (PON-1). Forty-five intact bitches were included in the study, being randomly distributed into three groups: laparoscopic OVE (L), midline OVE (M), and flank OVE (F). Serum CRP, Hp, Alb, and PON-1 were measured before surgery, 1, 24, 72, and 168 h post-intervention. CRP levels increased significantly 24 h post-surgery in the M and F groups, but no significant variation was observed in the L group at any time of the study period. Hp was significantly higher in group L than in group F 72 h post-surgery. Alb and PON-1 showed no statistical difference among groups or among sampling periods. CRP response suggests that the use of laparoscopic procedures produce lower inflammation compared to open conventional approaches when performing OVE in the bitch.

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.