The association of resveratrol and AFPI did not enhance the cryoresistance of ram sperm.

Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant.

Use of meloxicam with or without dipyrone in non-surgical embryo recovery in hair sheep: Effects on animal welfare.

The aim of this study was to determine the effectiveness of meloxicam with or without dipyrone on the welfare of ewes subjected to non-surgical embryo recovery (NSER). Two studies were carried out using 51 multiparous Santa Inês ewes. All animals received a standard oestrous synchronization treatment and a superovulatory protocol. In Study 1, 12 ewes received meloxicam (GM) before cervical transposition (1 mg kg-1 , i.v.), repeated 24 h after (1 mg kg-1 , i.m.), while the other 10 received a saline solution, remaining as a control group (GC1). In Study 2, ewes were allocated into a group of 15 ewes treated as GM of Study 1 associated with dipyrone (GMD; 50 mg kg-1 , i.m.) before cervical transposition, 12 h, and 24 h after, or a control group (GC2) of 14 ewes treated with saline solution. In both studies, heart and respiratory rates (RR), cortisol, glucose, total proteins, albumin and globulins blood concentration were recorded before sedation (BS), after sedation (AS), after cervical transposition, immediately after collection (IAC), and 0.5, 1.5, 3, 6, 12, 24 and 48 h after embryo collection (hAC). In Study 1, RR tended to be greater in GC1 (p = .08), serum total proteins and globulins values were lower and serum albumin values were greater in this group than GM (p = .003, p < .0001, and p < .0001, respectively). In Study 2, treatment of GMD tended to reduce the glycaemia at AS (p = .052) and reduced it at 3hAC (p < .0001), and 6hAC (p = .03). It also tended to reduce cortisol concentrations (p = .10). The other variables varied with NSER without interaction with the experimental treatments. In conclusion, in this study condition, NSER in sheep induced transient changes indicative of stress and possibly pain, therefore, affecting animal welfare. The administration of meloxicam was ineffective to reduce those responses, and the association of dipyrone had only slight effects without modifying the main welfare indicative responses in ewes subjected to NSER.

Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

Biostimulation with the ram effect increases the follicle recruitment, ovulatory diameter, and embryo viability rate in superovulated ewes.

The aim of this study was to compare the preovulatory follicular development and superovulatory outcomes in superovulated ewes which were either stimulated or not stimulated by placement with rams. The treatment regimen to super-stimulate ovarian follicular development was imposed to 28 ewes on "Day 0", from which 14 were stimulated with active rams for 48 h, starting at the time of the fifth FSH dose, with the ram being removed from the pen with the ewes and replaced which other ram every 12 h, using four different rams (group GRE). The other 14 ewes remained isolated from rams throughout the protocol (group GCON). All ewes were administered 133 mg of FSH, into six doses in decreasing quantities, every 12 h. The follicular development and number of ovulations were determined using ultrasonography. Biostimulation resulted in an increased number of large follicles, follicle diameter, and embryo viability rate (viable embryos/recovered structures∗100) was greater in ewes of the GRE than GCON group. The number of corpora lutea, follicular cysts, recovered structures, viable embryos, and degenerated and unfertilized structures was similar in ewes of the GRE and GCON group. Structures were recovered from more GRE than GCON ewes. In conclusion, biostimulation with rams during the last phase of the treatment regimen to induce superovulation enhanced the follicular growth and increased the embryo viability rate in ewes.

Hormonal protocol used for cervical dilation in ewes does not affect morphological embryo quality but reduces recovery rate and temporarily alters gene expression.

BACKGROUND: Information on the impact of hormonal protocols for cervical dilation on the quality of ovine embryos is scarce. METHODS: To compare the quality of embryos after cervical dilation protocol, ewes (n = 64) were allocated into either a treated group (100 µg estradiol benzoate intravenous and 0.12 mg cloprostenol intramuscularly, 12 hours before embryo collection plus 100 iu oxytocin intravenous 15 minutes before the collection procedure) or a control group (saline). Luteal function was analysed using ultrasonography and P4 measurement. Some collected embryos were frozen/thawed for gene expression, others were cultured in vitro, frozen/thawed for gene expression, and the remaining embryos were fixed for the apoptosis test (TUNEL test). RESULTS: The treatment reduced fluid (p=0.04) and structure (p=0.03) recovery rates, but the morphological quality, development stage, and apoptosis incidence of the embryos were not affected by treatment. The corpora lutea of the control group had greater blood perfusion (p = 0.002) and greater P4 concentrations at 6, 9, and 12 h after the treatment (p < 0.0001). The expression of BAX, BCL2, PRDX1, and HSP90 genes were not affected by the treatment. However, the embryos in the treated group had fewer NANOG and OCT4 transcripts than control embryos (p = 0.008; p = 0.006, respectively). After culture, there was no difference between the groups in any gene. CONCLUSION: The hormonal protocol for cervical dilation reduced the efficiency of embryo collection. In addition, the treatment induced luteolysis and a transient alteration of embryo gene expression, however there were no detectable changes in embryo morphological quality, development stage, or incidence of apoptosis.

Vaginal cytology and cervical mucus as tools to predict ovulation time in small ruminants.

The possibility of using cervical mucus and vaginal cytology as tools to predict ovulation time was assessed in 11 ewes and 11 does raised under tropical conditions. Every 12 h from progesterone removal to ovulation, estrus behavior, cervical mucus, vaginal cytology, and ovarian ultrasound exams were performed. In goats, vaginal cytology had 88% of accuracy on detecting the ovulation time. However, in sheep, there was no cell pattern in the vaginal cytology and cervical mucus varied at ovulation. In conclusion, both vaginal cytology and mucus evaluation may be useful tools to determine the ovulation time in goats; however, both strategies are less accurate in sheep.

Transcervical vs. laparotomy embryo collection in ewes: The effectiveness and welfare implications of each technique.

This study assessed animal welfare in ewes subjected to transcervical (TC) or laparotomy (LP) embryo collection, and the efficiency of these two techniques. Santa Inês ewes (n = 57) received a protocol for estrus synchronization and superovulation. Cervical dilation protocol was initiated 12 h before embryo collection in all ewes. Depending on the success of cervical passage, the embryos were collected from ewes by either TC or LP. Records were made of physiological (rectal temperature (RT) and heart rate (HR)), endocrine (cortisol concentration), biochemical (glycaemia, total proteins, globulin and albumin concentrations), and behavioral variables. Data were recorded before fasting (BF) and sedation (BS), during (DC) and immediately after embryo collection (IAC), and 1 h (1hAC), 3 h (3hAC), 6 h (6hAC), 12 h (12hAC), 24 h (24hAC), and 48 h (48hAC) after embryo collection. The LP and TC procedures were applied to 22 and 35 ewes (with 100.0% and 94.3% of procedures being successful, respectively). The use of LP took longer than TC (P = 0.007) but was less effective in the recovery of uterine fluid and structures (P = 0.0002 and P = 0.0180, respectively), with no difference in the number of viable embryos recovered per animal. The TC procedure induced a greater RT at DC (P = 0.002) and IAC moments (P < 0.0001). The heart rate was greater in TC than LP in IAC (P = 0.036). On the other hand, HR was greater with LP at 12hAC (P = 0.033) and 24hAC (P = 0.002). There was no interaction between the procedures and time on total proteins, albumin, or globulin concentrations. The TC procedure induced greater glycaemia than LP in IAC (P < 0.0001). LP induced greater serum cortisol concentration than TC at DC, IAC, 1hAC (P = 0.0004; P = 0.0006; P = 0.036, respectively), even though it was greater in the TC than the LP procedure at 3hAC (P = 0.008). In conclusion, the TC embryo collection was more effective than the traditional LP procedure. Although both embryo collection procedures affected ewes' welfare, the TC procedure is probably less stressor than the LP.

Successful transcervical uterine flushing can be performed without or reduced dose of oestradiol benzoate in cervical relaxation protocol in Dorper ewes.

This study assessed the efficiency of cervical relaxation protocol using none, half or full dose (1.0 mg) of oestradiol benzoate in Dorper ewes subjected to non-surgical embryo recovery (NSER). Thirty-six pluriparous ewes received progestogen sponge (60 mg) for 9 days plus eCG administration (300 IU i.m.) 24 hr before sponge removal. Ewes were not mated and were randomly assigned to receive at 16 hr before NSER 37.5 µg d-cloprostenol i.m. and different doses of oestradiol benzoate: 0.0 mg (0EB group; n = 12); 0.5 mg (0.5EB group; n = 12) or 1.0 mg of oestradiol (1.0EB group, n = 12). All ewes received oxytocin (50 IU) i.v. 20 min before NSER, which was performed 8 days after sponge removal. Corpora lutea were counted by transrectal ultrasonography 24 hr before NSER. After procedure, the ewes were kept in natural breeding period to check their post-NSER fertility. NSER was performed in 91.7% (33/36) of the animals with overall fluid recovery efficiency over 97% (p > .05). The cervical transposing with Hegar dilator was longer (p < .05) in 0EB (4.2 ± 0.3 min) compared to 0.5EB (1.7 ± 0.3 min) and 1.0EB group (1.5 ± 0.3 min). The cervical transposing with mandrel/catheter was longer (p < .05) in 0EB (2.4 ± 0.5 min) than 1.0EB group (1.3 ± 0.5 min). Overall duration of uterine flushing was 25.4 min with structure recovery rate of 43.5%, with no difference among groups (p > .05). The post-NSER fertility was higher (p < .05) in 0.0EB (90%) than 0.5EB group (36.4%). In conclusion, NSER can be successfully performed in Dorper ewes by using a cervical relaxation protocol without oestradiol benzoate.

Pregnancy rate after fixed-time transfer of cryopreserved embryos collected by non-surgical route in Lacaune sheep.

This study investigated the feasibility of applying fixed-time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non-surgically recovered from superovulated donors (n = 39) on day 6-7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed-time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non-surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.

Efeito da técnica de coleta e do estágio do ciclo estral na recuperação de oócitos de boa qualidade em felinos e caninos domésticos / The effect of recovery technique and stage of estrous cycle on the recoveryof good quality oocytes in domestic felines and canines

A obtenção de oócitos de boa qualidade é essencial para o sucesso de diversas biotécnicas reprodutivas. Objetivou-se determinar o efeito de duas técnicas na recuperação de oócitos de boa qualidade em gatas e cadelas em diferentes estágios reprodutivos. Foram utilizados 43 pares de ovários de gata e 35 de cadela após realização da ovariosalpingohisterectomia eletiva. A fase do ciclo estral foi classificada em inativa, folicular ou luteal. Os ovários da fase folicular foram divididos em três grupos: PUN) punção dos folículos com agulha; PUN+FAT) fatiamento do mesmo ovário já puncionado; e FAT) fatiamento do segundo ovário. Os ovários das fêmeas em fase luteal e inativa foram submetidos ao FAT. Foram obtidos no total 974 oócitos (~23/animal) nas fêmeas felinas e 940 (~27/animal) nas caninas. O fatiamento recuperou número superior (P0,05) entre as técnicas de coleta na qualidade de estruturas recuperadas. A quantidade de oócitos recuperados em cada fase foi similar (P>0,05). Contudo, a fase inativa foi superior à luteal (P<0,05) e semelhante à folicular na quantidade de oócitos de boa qualidade em gatas e não houve diferença em cadelas. Conclui-se que o fatiamento recupera maior quantidade de oócitos, não influenciando em sua qualidade. As fases inativa e folicular recuperam maior quantidade de oócitos de boa qualidade em gatas e não afetam a recuperação em cadelas. Portanto, para otimizar o uso das biotecnologias, deve-se levar em consideração o estágio do ciclo estral em fêmeas felinas e a técnica de coleta utilizada na recuperação de oócitos.

The recovery of good quality oocytes is essential for the success of various reproductive biotechniques. The aim of this study was to determine the effect of two techniques on the recovery of good quality oocytes in queens and bitches at different reproductive stages. A total of 43 pairs of ovaries of queens and 35 of bitches after elective ovariosalpingohisterectomy were performed. The estrous cycle phase was classified as inactive, follicular or luteal. The ovaries of the follicular phase were allocated into three groups: PUN) puncture of the follicles with a needle; PUN + SLI) slicing of the same ovary already punctured; and SLI) slicing of the second ovary. The ovaries of luteal and inactive females were submitted to SLI. A total of 974 oocytes (~23/animal) were obtained in feline females and 940 (~27/animal) in canines females. The SLI technique recovered superior number (P0.05) between the collection techniques in the quality of recovered structures. The number of oocytes recovered in each phase was similar (P>0.05). However, the inactive phase was higher than luteal (P<0.05) and similar to the follicular phase in the quantity of good-quality oocytes in queens and there was no difference in bitches. In conclusion, it is preferable to perform the slicing technique to recover more oocytes in both species. Moreover, in queens it is possible to obtain good quality oocytes in the inactive phase and in bitches the estrous cycle phase does not influence the quality.

Efeito da técnica de coleta e do estágio do ciclo estral na recuperação de oócitos de boa qualidade em felinos e caninos domésticos / The effect of recovery technique and stage of estrous cycle on the recovery of good quality oocytes in domestic felines and canines

A obtenção de oócitos de boa qualidade é essencial para o sucesso de diversas biotécnicas reprodutivas. Objetivou-se determinar o efeito de duas técnicas na recuperação de oócitos de boa qualidade em gatas e cadelas em diferentes estágios reprodutivos. Foram utilizados 43 pares de ovários de gata e 35 de cadela após realização da ovariosalpingohisterectomia eletiva. A fase do ciclo estral foi classificada em inativa, folicular ou luteal. Os ovários da fase folicular foram divididos em três grupos: PUN) punção dos folículos com agulha; PUN+FAT) fatiamento do mesmo ovário já puncionado; e FAT) fatiamento do segundo ovário. Os ovários das fêmeas em fase luteal e inativa foram submetidos ao FAT. Foram obtidos no total 974 oócitos (~23/animal) nas fêmeas felinas e 940 (~27/animal) nas caninas. O fatiamento recuperou número superior (P<0,05) de oócitos. Não houve diferença (P>0,05) entre as técnicas de coleta na qualidade de estruturas recuperadas. A quantidade de oócitos recuperados em cada fase foi similar (P>0,05). Contudo, a fase inativa foi superior à luteal (P<0,05) e semelhante à folicular na quantidade de oócitos de boa qualidade em gatas e não houve diferença em cadelas. Conclui-se que o fatiamento recupera maior quantidade de oócitos, não influenciando em sua qualidade. As fases inativa e folicular recuperam maior quantidade de oócitos de boa qualidade em gatas e não afetam a recuperação em cadelas. Portanto, para otimizar o uso das biotecnologias, deve-se levar em consideração o estágio do ciclo estral em fêmeas felinas e a técnica de coleta utilizada na recuperação de oócitos.

The recovery of good quality oocytes is essential for the success of various reproductive biotechniques. The aim of this study was to determine the effect of two techniques on the recovery of good quality oocytes in queens and bitches at different reproductive stages. A total of 43 pairs of ovaries of queens and 35 of bitches after elective ovariosalpingohisterectomy were performed. The estrous cycle phase was classified as inactive, follicular or luteal. The ovaries of the follicular phase were allocated into three groups: PUN) puncture of the follicles with a needle; PUN + SLI) slicing of the same ovary already punctured; and SLI) slicing of the second ovary. The ovaries of luteal and inactive females were submitted to SLI. A total of 974 oocytes (~23/animal) were obtained in feline females and 940 (~27/animal) in canines females. The SLI technique recovered superior number (P<0.05) of oocytes. There was no difference (P>0.05) between the collection techniques in the quality of recovered structures. The number of oocytes recovered in each phase was similar (P>0.05). However, the inactive phase was higher than luteal (P<0.05) and similar to the follicular phase in the quantity of good-quality oocytes in queens and there was no difference in bitches. In conclusion, it is preferable to perform the slicing technique to recover more oocytes in both species. Moreover, in queens it is possible to obtain good quality oocytes in the inactive phase and in bitches the estrous cycle phase does not influence the quality.

Reproductive features and use of an anti-inflammatory drug in estrus-induced dairy goats artificially inseminated in a standing position with cervix immobilization.

This study evaluated reproductive features and role of Flunixin-Meglumine at timed artificial insemination (AI), using a new technique of standing position with cervix immobilization. In Experiment 1, 10 goats (n=5 nulliparous [Null] and 5 pluriparous [Plu]) were evaluated after estrus induction by recorded reproductive parameters to define the ideal time for AI. In Experiment 2, goats were artificially inseminated 51-54h after sponge removal with frozen-thawed semen. At AI, 1mL saline (CONTROL; 18 Null and 14 Plu) or 50mg Flunixin-Meglumine (FLUNIXIN; 15 Null and 18 Plu) was administered i.m. Location of semen deposition was recorded for both groups. In Experiment 1, all sexual behavior and ovulatory parameters were similar between Null and Plu for estrus response and ovulation (100%), interval from sponge removal to ovulation (∼64.2h), largest ovulatory follicle diameter (∼6.6mm), and number of ovulations (∼2.0). In Experiment 2, pregnancy rate was superior (P<0.01) for CONTROL (62.5%; 10 Null and 10 Plu) than FLUNIXIN (30.3%; 3 Null and 7 Plu) goats. Regardless of the treatment, intrauterine AI was more frequent (P<0.01) in Plu (100.0%; 32/32) than in Null (69.7%; 23/33) goats. Moreover, AI was more time-consuming (P<0.01) in Null (44±37s; 4-139s) than in Plu (21±19s, 4-78s) goats. Therefore, administration of Flunixin-Meglumine at the time of AI adversely affected pregnancy rate. High rates of intrauterine cervical penetration were obtained, achieving good pregnancy rates in goats not receiving Flunixin-Meglumine.