The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus.

BACKGROUND: Mycoplasma bovis is an important pathogen for the cattle industry worldwide causing significant economic losses. Several transmission routes, including those related to reproduction, have been described. Indeed, the pathogen can colonize the female reproductive tract after artificial insemination (AI) with contaminated semen. Lactobacillus spp.-based probiotics have been used for vaginal dysbiosis treatment in women and cows although their role in controlling cervico-vaginal infections due to M. bovis is unknown. The objective of the present work is to assess the viability of M. bovis (PG45, NCTC 10131) in experimentally contaminated cervical mucus after the addition of Lactobacillus spp. at different concentrations as a competing agent and pH acidifier. RESULTS: The addition of probiotic at a concentration higher than 108 colony forming units (CFU/mL had a detrimental effect (P < 0.05) on mycoplasma viability in cervical mucus. This coincided with a significant LAB growth and an important decrease in pH from 8.4 to 5.6 (P < 0.05). However, after the addition of less concentrated probiotic, M. bovis survival was not affected and there was no significant LAB growth despite the drop of pH from 8.4 to 6.73 (P < 0.05). CONCLUSION: The addition of concentrations higher than 108 CFU/mL of Lactobacillus spp. negatively affects M. bovis viability in bovine cervical mucus under in vitro conditions. Although the effect observed on the pathogen viability seems to be related to the pH decrease after LAB proliferation in cervical mucus, further studies are necessary to elucidate if other factors are implicated. Nevertheless, the administration of Lactobacillus spp.-based probiotics might be used in the future to control M. bovis proliferation in the cervico-vaginal tract of cows.

Effect of exogenous progesterone administration on luteal sensitivity to PGF during the early development of the corpus luteum in mares and cows.

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.

Use of a split or single prostaglandin F(2α) treatment in a 6-day synchronization protocol for nonlactating dairy cows.

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.

Effect of the bovine oviductal fluid on in vitro fertilization, development and gene expression of in vitro-produced bovine blastocysts.

Oviductal microenvironment generally provides better conditions for early embryo development than the conventional in vitro system. In an attempt to simulate the oviduct conditions or the main potentially influencing factors, the effect was studied of a bovine oviductal fluid (bOF) treatment applied prior to IVF on (i) IVF parameters, (ii) cleavage rate, (iii) blastocyst yield and (iv) blastocyst quality. Embryo quality was assessed by morphological embryo quality and relative transcript abundance of several developmental genes in bovine blastocysts. Furthermore, to study the effect of bOF without the male effect and zona-sperm interaction, artificially activated metaphase II oocytes were also treated with bOF. In vitro-matured bovine oocytes from abattoir ovaries were treated or untreated with bOF for 30 min and then washed prior to IVF or activation. Subsequently, in vitro-fertilized and parthenogenetic 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 IVF bovine embryos and neither on morphological quality of IVF blastocysts. G6PD and SOD2 genes from IVF blastocysts showed significant changes in their expression after a bOF treatment. Significant differences were found for the expression of SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes between excellent or good blastocysts (Grade 1) and fair blastocysts (Grade 2). To our knowledge, this is the first study that evaluates the effect of bOF oocyte treatment on fertilization parameters, development and quality of bovine embryos.

Effects of d-cloprostenol dose and corpus luteum age on ovulation, luteal function, and morphology in nonlactating dairy cows with early corpora lutea.

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 <5d old. To date, it is not known whether a larger dose will have a more potent luteolytic effect in cows during early diestrus. The objective of this study was to characterize the effect of 2 doses of d-cloprostenol (150 and 300 µg) on the progesterone concentration, luteal diameter, and ovulation rate in nonlactating dairy cattle 96 to 132 h postovulation. Twenty nonlactating dairy cows were included in the study. Each cow received 2 treatments of d-cloprostenol in 2 consecutive cycles: a standard dose of 150 µg and a double dose of 300 µg. The cows were allocated randomly to 1 of 4 groups (5 cows in each group) according to the age of the CL at the time of treatment: 96, 108, 120, and 132 h. The exact time of ovulation was known within 12h, because of twice per day ultrasound examination. The CL diameter and progesterone concentration were measured before treatment (d 0) and 2 and 4d after treatment. Within each CL age group, the effect of d-cloprostenol dose on luteolysis was determined. More cows treated with double dose tended to have full luteolysis compared with the standard dose (8/10 vs. 4/10, respectively). This effect was only apparent in cows with CL of 120 and 132 h but not in earlier CL. The interval from treatment to ovulation was shorter (3.3 ± 0.1d) in cows treated with a double dose than in cows treated with the standard dose (4.5 ± 0.4d).

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs.

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.

The effect of a single high dose of PGF2α administered to dairy cattle 3.5 days after ovulation on luteal function, morphology, and follicular dynamics.

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%.

Sry-negative XX sex reversal in a French Bulldog.

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.

Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT.

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.

Intracytoplasmic sperm injection in livestock species: an update.

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.

The use of R-roscovitine to fit the 'time frame' on in vitro porcine embryo production by intracytoplasmic sperm injection.

Micromanipulation of oocytes is time consuming during ICSI experiments; however the 'time frame' to manipulate oocytes without a drop in efficiency is not very wide due to the use of not completely matured and/or aged MII oocytes. Therefore, the aim of this work was to study the effect of a short roscovitine pretreatment for 5 h and two different IVM periods (5R + 40IVM or 5R + 45IVM) and a prolonged IVM time from 45 h (45IVM) to 50 h (50IVM) on parthenogenetic and ICSI embryo development, in order to fit the time frame to manipulate pig oocytes to the whole labour day session. In the first experiment, oocytes, pretreated with roscovitine and IVM cultured for 5 h, showed a similar nuclear stage as non-cultured oocytes and a significantly higher percentage of GVI-GVII oocytes compared with non-roscovitine treated oocytes cultured for 5 h in IVM conditions. When COC were cultured under the 5R + 40IVM system, nuclear maturation and cleavage rates after electrical activation were significantly lower than when COC were cultured under the 45IVM, 50IVM and 5R + 45IVM culture systems (54.2% vs. 72.6-76.8% and 58.8% vs. 81.4-88.3%, respectively). However, this difference was not statistically significant for parthenogenote blastocyst rate. No differences were observed in MII and in parthenogenote and ICSI embryo development among 45IVM, 50IVM and 5R + 45IVM experimental groups. In conclusion, under our conditions and using parthenogenetic and ICSI embryos, we observed that it is feasible to prolong the pig oocyte manipulation 'time frame' by at least 5 h with no significant drop in blastocyst rate.