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.

Reproductive seasonality of hair rams under tropical conditions: an alternative for non-seasonal lamb production?

Reproductive seasonality limits the periods of breeding on the year and, therefore, productive output. However, some breeds appear as probably non-seasonal. The aim of the study was to characterize the seasonal pattern of Santa Inês rams, including an ultrasound characterization of the reproductive tract, testosterone concentrations, and semen characteristics. Fifteen Santa Inês rams remained in a grazing system with concentrate supplementation, and measurements of the reproductive tract and ultrasound evaluation (biometrics and pixel intensity) of the testicles and accessory sex glands were monthly recorded. Computerized seminal evaluations were also performed monthly, and serum testosterone concentration was measured every 15 days. Body weight and condition remained stable throughout the year. In general, reproductive traits varied along the year and reached maximum values during autumn and minimum in spring. Despite that, as fresh semen remained with enough quality to breed all along the year, seasonality does not appear as a limiting factor to breed along the year. Therefore, Santa Inês rams can be used for all-year-round breeding or for crossbreeding when rams from other breeds decrease their fertilizing ability.

Characterization of uterine involution using B-mode ultrasonography, color Doppler and elastography (acoustic radiation force impulse) for assessing postpartum in Santa Inês ewes.

The aim of this study was to investigate uterine involution using ultrasonography techniques during postpartum. Postpartum ultrasonography evaluation (B-mode, color Doppler and Acoustic Radiation Force Impulse elastography) of the uterus was performed by transabdominal approach at immediate after birth and sequentially every 48 hours, during 30 days. The uterine echotexture did not present significant variations (P >0.05) being homogeneous in most evaluations; echogenicity of the uterus increased along the evaluation period (P =0.0452). Progressive and remarkable decrease of the total uterine diameter (UD) were observed (P <0.0001), especially during the first days postpartum. The thickness of uterine wall gradually decreased, as well the endometrial, myometrium and lumen diameters (P <0.0001). Uterine blood flow was assessed by Doppler and decreased during postpartum period, being significantly lower (P=0.0225) on the 30th day of postpartum. Uterine parenchyma presented as homogeneous dark areas (not deformable) on qualitative ultrasound elastography and the means shear velocity values of the uterine wall on quantitative elastography did not differ. This is the first study that evaluate the stiffness of uterine wall in healthy ewes, providing baseline data about quantitative and qualitative stiffness of the normal uterus, and it may be a useful tool for early diagnosis of uterine alterations during the postpartum period, using the reference parameter established for the assessment of uterine integrity during postpartum period.

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.

Influence of eCG and reproductive management in the resynchronization of ovulation in dairy goats.

Resynchronization protocols have been proposed as a way of shortening females' unproductive time in the flock, with good results in cattle and sheep. In goats, initial studies have shown that a second progestogen device inserted before luteolysis and pregnancy diagnosis does not interfere with the corpus luteum lifespan or functionality. This study aimed to evaluate the follicular growth, ovulation pattern and pregnancy rate after insertion of a second and new progestogen device for resynchronizing, with or without equine Chorionic Gonadotrophin (eCG), submitted to natural mating (NM) or artificial insemination (AI) to propose a viable resynchronization protocol for dairy goats. A total of 38 multiparous Saanen goats underwent a short-term progesterone protocol [six days exposed to medroxyprogesterone acetate (MAP) intravaginal sponges + 200 IU eCG and 0.12 mg of cloprostenol sodium on the 5th day + 0.025 mg of lecirelin 34 hours after sponge withdrawal] and, on day 16th after the ovulation, received a new MAP device which was retained until day 21. At this moment females were split into four groups: GeCG+NM - 100 IU eCG with NM; GSal+NM - saline solution with NM; GeCG+AI - 100 IU eCG with AI; and GSal+AI - saline solution with AI. Ultrasound scans were performed every 12 h from sponge withdrawal (day 21) until 108 h after sponge withdrawal (day 25) for follicular dynamics evaluation, at 240 h (day 31) for assessing the presence of active corpus luteum, and on day 60 for pregnancy diagnosis. No differences were found regarding ovulation time, synchronization and follicle size. However, GeCG+NM presented a greater estrus manifestation rate (100%) and pregnancy rate (62.5%) when compared to GSal+AI. In conclusion, resynchronization protocols in dairy goats may present satisfactory results.

Administration of butorphanol with ketamine/xylazine sedation reduces the negative responses to electroejaculation in rams.

This study aimed to identify the possible advantages of administering butorphanol (BUT), associated with ketamine/xylazine (KX) sedation or not, for semen collection using electroejaculation (EE) in rams. Fifteen rams received four treatments in a cross-over design using a 2 × 2 factorial arrangement. The treatments were 1) control; 2) received KX; 3) received BUT; and 4) received both treatments. The responses to the procedures regarding heart rate, respiratory frequency, and rectal temperature were recorded, glucose and creatine kinase were measured, and semen quality was determined. More pulses were required when BUT was administered (P = 0.0067), but rams vocalized fewer times (P = 0.046). The administration of KX also tended to reduce the respiratory frequency (P = 0.068) and rectal temperature (P = 0.089), with no other effects. The administration of BUT reduced the heart rate (P < 0.0001), and there was an interactive effect between the administration of BUT and time on the heart rate (P < 0.0001), respiratory frequency (P = 0.01), and rectal temperature (P = 0.047). The administration of BUT reduced the heart rate increase immediately after EE (P < 0.0001). The respiratory frequency following EE was greater when BUT was administered (P < 0.0001), but the administration of BUT reduced the rectal temperature at the same time (P = 0.002). Glucose concentration was greater when BUT was administered (P < 0.0001). The only significant effect on the ejaculate characteristics was from the interaction between KX and BUT on sperm concentration (P = 0.004). The administration of KX alone increased sperm concentration (P = 0.035), but when BUT was added to KX, sperm concentration decreased (P = 0.002). Moreover, sperm concentration was greater when only BUT was administered than when KX and BUT were administered simultaneously (P = 0.037). The addition of BUT markedly decreased most negative responses more effectively than sedation with KX alone. In conclusion, BUT appears to be an interesting alternative to reduce the welfare concerns raised by the use of EE in rams; however, its possible effects on the ejaculate characteristics when associated with other anesthesia/sedation drugs require further study.

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.

Assessment of dopperfluxometric indices of maternal-fetal structures in pregnant ewes.

The aim of this study was to evaluate the blood flow of the uterine artery, fetal aorta and umbilical artery in the physiological pregnancy of sheep by means of pulsed Doppler throughout the gestational period. Thirty Santa Inês ewes weighing between 45.4±4.3 kg and aged 2 to 5 years were selected. The evaluations were carried out weekly from the 3rd to the 21st gestational week. Peak systolic velocity (PSV), end diastolic velocity (EDV) and resistance index (RI) were obtained. Analysis of variance was performed, and the minimum significant comparison of means was obtained by the BH test with adjusted P<0.05. The results were expressed as mean ± standard error. For the fetal aorta, there was an increase in the EDV values and a decrease in the PSV and RI throughout pregnancy. For the uterine artery, PSV and EDV did not present significant variation, whereas the RI showed a reduction in the last week. Increased EDV values were found for the umbilical artery throughout pregnancy. For the PSV there was no significant difference, as the RI was reduced at the end of pregnancy. The results obtained are expected to contribute to a more complete understanding of the hemodynamic changes resulting from pregnancy.

Ultrasonographic characteristics of the testes, epididymis and accessory sex glands and arterial spectral indices in peri- and post-pubertal Nelore and Caracu bulls.

Ultrasonography can provide information about the integrity of organs; however, rarely is applied to the reproductive organ evaluation of bulls. The objective of the present study was to characterize and compare values for variables and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as spectral Doppler indices of the testicular and internal iliac arteries, between peri- and post-pubertal Nelore and Caracu bulls. Nelore (n = 203) and Caracu (n = 79) bulls were assigned by age class: peri-pubertal (12-15 months) and post-pubertal (> 22 months). Data were analyzed using SAS's PROC MIXED procedure (P < 0.05). The biometric variables of the testes and cauda epididymis differed between peri- and post-pubertal Nelore and Caracu bulls. There was a difference between breeds for the vesicular glands, ampulla of vas deferens, disseminate portion of the prostate, and craniocaudal dimension of the bulbourethral glands. Echogenicity of the testicular parenchyma differed between breeds and age classes. The pulsatility and resistive indices of the testicular arteries differed between Nelore and Caracu bulls. The biometric and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as of the arterial indices in bulls are affected by genetic group and age class, and when assessed there is useful information regarding the progression of sexual maturation.

B-mode ultrasonography and ecobiometric parameters for assessment of embryonic and fetal development in sheep.

The aim in this study was to evaluate the maternal-fetal development during physiological gestation in sheep by means of ultrasonography and to predict the gestational age by mathematical formulae. Thirty Santa Inês sheep were included. Evaluations were performed weekly from the 3rd to 21st gestational week. The Shapiro test was used to determine normality of the data, and Spearman test was used for determining correlations of real or transformed measurements as gestation advanced. If there was a significant difference detected, variable adjustments and gestational weeks were tested using regression models. Significance level was set at 5% and the results presented as mean ± SD. Principal components analysis (PCA) was used for data clustering and graphic analysis. The gestational vesicle, abdominal, thoracic, biparietal, ocular orbit, and placentome diameters; and the lengths of crown-to-rump, nuchal to snout, scapula, humerus, radius, metacarpus, femur, tibia, metatarsal, heart length, heart width and kidneys were measured. Using pulsed Doppler ultrasonography, heart rate (HR) was obtained. The greatest coefficients of determination (r²) were obtained for the biparietal diameter (BPD; 96.5%) and metacarpus length (MCAR; 93%), followed by the abdominal diameter (AD; 91.7%) and heart width (HTW; 90.2%). The PCA analysis indicated graphically that at week 8 to 9, there was no significant change related to technique, whereas, at weeks 19, 20, and 21 a decrease in growth was observed, with the greatest data variability at week 19. Ultrasonographic assessments indicated there were significant correlations between developmental stages of all maternal-fetal structures and the gestational age, which can be used as reference values for gestational age assessments and detection of development abnormalities.

Corpus luteum dynamics after ovulation induction with or without previous exposure to progesterone in prepubertal Nellore heifers.

The objectives of this study were 1) to monitor corpus luteum (CL) dynamics after two different protocols of ovulation induction in prepubertal Nellore heifers, and 2) to determine differences in luteal function. Fifty-seven heifers (weight 289.61 ± 32.28 kg, BCS 5.66 ± 0.65, age 17.47 ± 0.81 months) were divided into two groups: GP4+GnRH received a progesterone (P4) device of 3rd use for 10 days, followed by the administration of 0.02 mg buserelin acetate (GnRH) 48 h after removal of the device, and GGnRH received only GnRH. The CLs formed were monitored by ultrasonography every 2 days until their functional regression (decrease in the color Doppler signal and serum P4 concentration < 1 ng/mL), determining their diameter and area, numerical pixel value (NPV), pixel heterogeneity, and vascularization percentage. The peak systolic velocity, end diastolic velocity, resistivity index and pulsatility index (PI) of the ovarian artery and serum P4 concentration were also measured. A lifespan of the CL of more than 16 days was classified as normal-function and of less than 16 days as premature regression. The variables were compared between treatments, CL categories (normal-functional, prematurely regressed or non-functional), days of evaluation, and their interactions using the MIXED procedure of the SAS program (p ≤ 0.05). Three animals of each group (6/57 = 11%) did not respond to treatment, corresponding to an ovulation rate of 89%. There was a higher percentage of normal-function CLs in GP4+GnRH (81%) and a higher percentage of non-functional CLs in GGnRH (52%; P4 concentration < 1 ng/mL in all assessments). Normal-function CLs exhibited a greater area, vascularization percentage and P4 concentration than prematurely regressed and non-functional CLs. Lower diameter, area, NPV and P4 concentration were observed for non-functional CLs, but there was no difference in vascularization percentage compared to prematurely regressed CLs. Progesterone concentration was efficient in diagnosing CL function and was positively correlated with CL area (r = 0.62; p < 0.001) and vascularization percentage (r = 0.38; p < 0.001). Diameter and PI were important for the early diagnosis of non-functional and prematurely regressed CLs, respectively. In conclusion, luteal function differed for the first CL that develops after ovulation induction in prepubertal heifers. Ultrasonographic parameters (diameter, area, NPV, vascularization percentage, and PI) can be used to predict CL function.