Review: Non-surgical artificial insemination and embryo recovery as safe tools for genetic preservation in small ruminants.

Artificial insemination (AI) and in vivo embryo production (or multiple ovulation and embryo transfer, MOET) programs are both instrumental in accelerating the propagation of genetically and economically superior goats and sheep. The aim of this review was to present the current gestalt of non-surgical AI and embryo recovery (NSER) procedures in small ruminants. Small body size, precluding rectal palpation, and highly limited penetrability of the uterine cervix in ewes are the major reasons for the scarce use of non-surgical assisted reproduction techniques in this species. As a result, AI and embryo recovery techniques in sheep mainly involve laparoscopy or laparotomy (LAP). In does, however, the Embrapa method of AI allows for successful intrauterine deposition of semen, resulting in pregnancy rates from 50 to 80% under field conditions (>3 000 goats inseminated) when frozen-thawed semen is used. After the administration of prostaglandin F2α (PGF2α), non-surgical (transcervical) embryo recovery is also feasible in goats, with the cervical penetration rate approaching 100%. There is a paucity of information on the efficacy of non-surgical AI using frozen semen in sheep, but the results are satisfactory with fresh, cooled, or chilled ram semen. An application of the NSER technique in ewes has greatly improved over the last decade, and cervical penetration rates of ∼90% can be achieved when a hormonal cervical dilation protocol using PGF2α, oxytocin, and/or estradiol ester (e.g., estradiol benzoate) is applied. In some genotypes of sheep, sufficient cervical dilation can be induced without estradiol ester included in the protocol. Several studies indicated that recovery of transferable quality ovine embryos using NSER is comparable to that employing a ventral midline laparotomy, and NSER is evidently a method of choice when animal welfare is concerned. Considering both the number of retrievable embryos and animal well-being, the NSER is a viable alternative for surgical procedures. With further developments, it has the makings of a primary, if not exclusive, embryo recovery technique in small ruminants worldwide.

Ovarian steroids modulate the systemic inflammatory response OF COWS challenged with lipopolysaccharide (LPS) intrauterine infusion.

Postpartum uterine infections of dairy cows promote a local and systemic inflammation and interfere with reproductive efficiency. The aim of this study was to evaluate the effect of steroid hormones including progesterone (P4) and estradiol (E2) on the systemic inflammatory response of cows after being challenged with an intrauterine infusion of lipopolysaccharide (LPS). For this, a hemogram and serum dosage of haptoglobin (Hp) in eight primiparous Gir cows ovariectomized were performed on day (day 0) and after 24 h (day +1). Four cows (n = 4) were challenged (day 0) with 20 mL of 0.9% NaCl + 12.5 µg/kg LPS, and four cows (n = 4) were challenged (day 0) with 20 mL of 0.9% NaCl. For this, the study was divided in four experimental groups as: (1) Control group: without any hormonal treatment before day 0; (2) Group 24 h - E2: 1 mg of estradiol benzoate 24 h before (day -1); (3) Group 24 h - P4: 2.0 g of P4 device 24 h before (day -1); (4) Group 14 d - P4: 2.0 g of P4 device 14 days before (day -14). In the systemic response to LPS, there was an increase in Hp (control group; 24 h - P4 group; 14 d - P4 group), and on day +1 the Hp of 14 d - P4 group was higher when compared to the other groups. On day 0, the 14 d - P4 group had an increase in circulating leukocytes and lymphocytes cells than the control group (P < 0.01). On day +1 after LPS-challenge the 14 d - P4 group showed a decrease in circulating lymphocytes, eosinophils, and monocytes (P < 0.05). A neutrophilia with left shift in the two treatments with P4 (day +1), in addition to a thrombocytopenia and lower platelets compared to the 24 h - E2 group (P < 0.05) (day 0) were recorded. It was concluded that ovariectomized cows challenged with LPS, previously submitted to steroid hormones induce a systemic inflammatory response. Also, the systemic response is more intense after previous prolonged exposure to P4 and less intense after exposure to E2. This study provided important information relating the effect of ovarian steroids on the systemic inflammatory response of cows challenged with intrauterine LPS.

Exogenous progestogens differentially alter gene expression of immature cumulus-oocyte complexes in sheep.

This study evaluated the role of progesterone (P4) and medroxyprogesterone acetate (MAP) on the molecular status of immature cumulus-oocyte complexes (COCs) and the implications for oocyte quality in sheep. The number of viable COCs per ewe and the rate of COCs screened for developmental competence by brilliant cresyl blue positive (BCB+) were similar (P > 0.05), respectively, across treatments (P4: 7.7 ± 0.7 and 4.7 ± 1.2; MAP: 5.7 ± 1.0 and 3.5 ± 2.3; and control: 5.7 ± 1.1 and 3.6 ± 2.4). The COCs' gene expression was altered by exogenous progestogens compared with the control group: markers of steroidogenic pathway (FSH receptor [FSHr], LH receptor [LHr], and estradiol receptor α) and of quality (zygote arrest 1, growth differentiation factor 9, and B-cell lymphoma 2) were in abundance in P4 (P < 0.05). In addition, reelin protein (RELN) was downregulated, and Bcl-2 was upregulated in MAP (P < 0.05). In the P4 vs MAP comparison, FSHr, LHr, and RELN genes were upregulated (P < 0.05) in the P4 group. In conclusion, P4 and MAP promoted dissimilar effects on transcriptome profiling of immature BCB-selected COCs, possibly due to the differences in the chemical structure of progestogens and concentrations of serum P4. Exogenous P4 impacted positively on the profile of genes related to oocyte quality.

Administration of a single dose of 300 IU of human chorionic gonadotropin seven days after the onset of estrus improves pregnancy rate in dairy goats by an unknown mechanism.

This study examined the effects of exogenous hCG administration on ovarian function and pregnancy rates in estrous-induced dairy goats during the transition into the breeding season. Eighty-six Toggenburg does received 60 mg of medroxyprogesterone acetate intravaginal sponge for 6 d plus 200 IU of equine chorionic gonadotropin and 30 µg of d-cloprostenol i.m. 24 h before sponge removal, and were then bred for 96 h. Seven days (D7) after first mating the does received either 1 mL of saline (the control group, n = 43) or 300 IU of hCG (the hCG-treated group, n = 43) i.m. Transrectal ovarian ultrasonography (B-mode and color Doppler) was performed on D7, D13, D17, and D21 and ultrasonographic pregnancy detection on D30. Pregnancy rate was higher (P < 0.05) in hCG-treated goats (90.7%; 39/43) than that in control animals (74.4%; 32/43). Accessory luteal structures (ALSs) were detected in 46.5% (20/43) of hCG-treated does. All hCG-treated does that had ALSs and 82.6% of goats without ALS post-treatment remained pregnant. The total luteal area increased (P < 0.05) from D7 to D13 in pregnant animals of both groups, whereas mean vascular area declined (P < 0.05) by D21 in all nonpregnant does. Serum progesterone concentrations increased (P < 0.05) on D21 in pregnant goats of both groups, but they were related to changes in luteal tissue content only in control does throughout the present study. Mean daily numbers of small- and medium-sized antral follicles decreased (P < 0.05) only in pregnant animals of both groups with a decline in medium follicle numbers occurring earlier in hCG-treated (D13) compared with control does (D17). To summarize, a single dose of hCG given on D7 after estrus was followed by a decrease in the number of medium-sized antral follicles in gestating hCG-treated does, induced the formation of ALSs in ~47% of all hCG-treated does, and significantly increased the pregnancy rate in estrous-induced Toggenburg goats in the transition to the breeding season.

Idade à puberdade e características reprodutivas de novilhas mestiças F1 Holandês x Gir com fenótipos divergentes para consumo alimentar residual / [Age at puberty and reproductive characteristics of F1 Holstein x Gyr crossbred heifers with divergent phenotypes for residual feed intake]

A relação da eficiência nutricional com a idade e o peso à puberdade e as características reprodutivas foram estudadas em 35 novilhas F1 Holandês x Gir, em ensaios usando o consumo alimentar residual (CAR) nos períodos pós-desaleitamento (PD) e pós-puberdade (PP). Novilhas PD (P<0,0001) e PP (P<0,001) foram ranqueadas como de alta, intermediária e baixa eficiências e apresentaram CAR de -0,259 -0,014 e 0,346kg/dia e -0,848 -0,096 e 0,842kg/dia, respectivamente. Novilhas PD diferiram (P<0,05) na idade à puberdade entre ranques de alta, intermediária e baixa eficiências, em média, aos 339,7; 346,4; 315,3 dias, respectivamente. Os pesos à puberdade de novilhas PD com alta, intermediária e baixa eficiências foram, em média, de 321±28,1kg e não diferiram (P>0,05) entre CAR. Não foi verificada correlação entre diâmetro folicular, taxa de crescimento do folículo, número de ondas foliculares no ciclo estral e taxa de gestação à primeira IA com o CARPD ou CARPP. Novilhas F1HG selecionadas para CAR com baixa eficiência podem ter idade à puberdade 10 dias a mais, quando comparadas às novilhas de alta eficiência para CAR. Não foi verificado que novilhas selecionadas para alta ou baixa eficiências alimentares, usando CAR, tivessem algum impacto negativo sobre características reprodutivas avaliadas.(AU)

The relationship between nutritional efficiency and age and weight at puberty and reproductive traits was studied in 35 F1 Holstein x Gyr heifers, in trials using residual food consumption (CAR) in the post-weaning (PW) and post-pubertal (PP) periods. PD heifers (P<0.0001) and PP (P<0.001) were classified as high, intermediate and low efficiencies and presented CAR of -0.259 -0.014 and 0.346kg/day and -0.848 -0.096 and 0.842kg/day, respectively. PD heifers differed (P<0.05) in age at puberty between high, intermediate and low efficiencies, on average, at 339.7, 346.4 and 315.3 days, respectively. The weight at puberty of PD heifers with high, intermediate and low efficiencies were, on average, 321±28.1kg and did not differ (P>0.05) between CAR. No correlation was found between follicular diameter, follicle growth rate, number of follicular waves in the estrous cycle and pregnancy rate at 1st AI with the CARPD or CARPP. F1HG heifers selected for CAR with low efficiency can be aged at puberty 10 days longer when compared to high-efficiency heifers for CAR. These results did not show that heifers selected for high or low food efficiency, using CAR, had negative impact on the reproductive characteristics evaluated.(AU)

Exogenous progestogen does not affect first-wave follicle populations and oocyte quality during ovarian stimulation with FSH in sheep.

The effect of short-term administration of medroxyprogesterone acetate (MPA) or natural progesterone (P4) during ovarian stimulation with FSH on oocyte recovery was investigated in Santa Inês ewes. Ewes were treated with an intravaginal sponge containing MPA for 6 d; GnRH was applied 36 h after sponge removal and FSH was given in 3 injections (40, 24, and 16 mg, respectively) every 12 h after (D0, approximate ovulation time). At the first FSH dose, the ewes received either a new MPA sponge (n = 10) or a controlled device for internal release impregnated with P4 (n = 10) or did not receive any device (n = 10). Ovarian dynamics were assessed every 12 h by transrectal ultrasonography from D-3 to D2. Oocytes were recovered by laparoscopic ovum pick-up (LOPU) on D2 and graded by morphologic quality. The number of small, medium, and large follicles at D0 and D2 (ultrasound examinations), number of both follicles aspirated and oocytes recovered at LOPU, recovery rate, and oocyte grade did not differ (P > 0.05) among treatments. Thus, the short-term use of MPA or P4 during ovarian stimulation did not affect the first-wave follicle population or morphologic quality of oocytes. We would suggest that, in this protocol, the use of exogenous progestin is unnecessary.

Influência da dificuldade de inseminação, temperamento e cortisol plasmático sobre a taxa de concepção de vacas e novilhas da raça Nelore inseminadas em tempo fixo / Influence of difficulty of insemination, temperament and plasma cortisol on conception rate of Nelore cows and heifers in timed AI

O objetivo do presente experimento foi avaliar efeito do estresse e da dificuldade de inseminação (DifIA) sobre a taxa de concepção (TC) de vacas (n=93) e novilhas (n= 72) Nelore submetidas à IATF. No D9, anotou-se nota de temperamento (NTe) e tempo da saída do brete (TSB) de todos os animais e coletou-se sangue das novilhas. No dia da IATF (D11), anotou-se NTe, TSB, DifIA e tempo de IA. A TC foi 36% para vacas e 46% para novilhas (P>0,05). Não foi observado efeito de Nte sobre TC (P>0,05). Porém, houve tendência para maior (P<0,10) TC nos animais que não apresentaram dificuldade de inseminação (DifIA1; TP=42%) em comparação aos animais com moderada ou alta dificuldade (DifIA2+DifIA3; TP=27%). Foi observado menor (P<0,05) tempo de IA para animais DifIA1 (17:31±06:02s) que animais DifIA2-3 (30:10±15:45s). Novilhas com maiores (P<0,05) níveis de cortisol apresentaram maior NTe (P<0,05). Entretanto, TC (59%) das novilhas menos reativas (cortisol=4,12±1,12ng/mL; NTe=3,2±0,6) não diferiu da TC (41%; P>0,05) das mais agitadas (cortisol=7,76±1,33ng/mL; NTe=3,82±0,79). Concluiu-se que avaliações de temperamento se relacionaram com nível de estresse, embora esses parâmetros não tenham afetado a TC deste trabalho. A maior dificuldade e/ou tempo necessário para se completar a IA demonstrou ser um potencial fator para a redução da fertilidade na IATF.(AU)

The objective was to evaluate the influence of stress and difficulty of insemination (DifIA) on conception rate (CR) of Nellore cows (n= 93) and heifers (n= 72) in Timed-AI. On D9, temperament (NTe) and time for chute exit (TSB) were recorded for all animals, and blood samples were colected from heifers. On the day of Timed-AI (D11), NTe, TSB, DifIA and time for AI were recorded. For cows, CR was 36% and for heifers 46% (P> 0.05). No effect (P> 0.05) of NTe was observed on CR. However, a tendency (P< 0,10) for higher CR was observed in animals with no difficulty for insemination (DifIA1; CR=42%) compared to animals that presented moderate or high difficulty (DifIA2+DifIA3, CR=27%). Time required for AI was lower (P< 0.05) in animals DifIA1 (17:31±06:02sec) than in animals DifIA2-3 (30:10±15:45sec). Heifers with greater (P< 0.05) cortisol levels presented higher Nte (P< 0.05). However, CR (59%) of less reactive heifers (cortisol=4,12±1,12ng/mL; NTe=3,2±0,6) did not differ from CR (41%; P> 0.05) of stressed animals (cortisol=7,76±1,33ng/mL; NTe=3,82±0,79). It was concluded that assessments of temperament were related to stress level, although these parameters did not affect the CR of this study. However, the higher difficulty and/or time to complete AI showed to be a potential factor for reducing fertility after timed-AI.(AU)

Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)

Use of human intravaginal tampon embedded with natural progesterone induces synchronous estrus in Santa Inês ewes / Uso de tampão intravaginal humano embebido com progesterona humana para indução e sincronização de estro em ovelhas Santa Inês

Este estudo avaliou a eficiência de três protocolos de indução de estro síncronizado em ovelhas da raça Santa Inês. Vinte e quatro ovelhas adultas foram equitativamente distribuídas em três grupos, de acordo com ordem de parto, peso corporal (kg) e escore da condição corporal. As ovelhas receberam implante vaginal de progesterona natural por seis dias mais 37,5µg de d-cloprostenol laterovulvar e 300UI de eCG i.m., 24 horas antes da remoção do dispositivo. Ovelhas controle receberam CIDR330mg de progesterona, e as demais receberam dispositivo absorvente intravaginal humano, tamanho mini, embebido com 200 (OB200) ou 400mg (OB400) de progesterona. Coletas de sangue foram feitas nos momentos D0 (antes da inserção dos dispositivos), D0+6h e diariamente, até um dia após retirada do dispositivo (D7). A progesterona (ng/mL) foi semelhante (P>0,05) em todos os tratamentos ao longo do período experimental, exceto no dia da remoção do dispositivo, quando as ovelhas controle (2,5±0,3) tiveram progesterona superior (P<0,05) às ovelhas OB200 (0,6±0,1) e OB400 (1,2±0,4). O percentual de animais em estro (100,0, 62,5 e 100,0%) foi semelhante (P>0,05) e o intervalo para o estro (46,3±3,9a, 26,4±4,5b e 31,2±5,8a,b) foi diferente (P<0,05) entre os animais dos grupos controle, OB200 e OB400, respectivamente. A taxa de concepção das ovelhas foi de 50,0% (4/8) para cada grupo (P>0,05). Dispositivos humanos e fonte de progesterona podem ser usados para induzir o estro sincronizado em ovelhas Santa Inês.(AU)

Hydrometra in dairy goats: Ultrasonic variables and therapeutic protocols evaluated during the reproductive season.

Hydrometra is characterized by the accumulation of fluid within the uterus due to the persistence of corpus luteum. The diagnosis of this disorder occurs with an ultrasonic exam. This study evaluated uterine drainage and fertility rates in goats after the use of d-cloprostenol in association or not with Gonadotropin-releasing hormone (GnRH) treatment. Twenty Saanen goats, diagnosed with hydrometra, received three 37.5-µg doses of d-cloprostenol laterovulvarly at 10-day intervals. On D5, the goats were assigned into two groups receiving 1 mL of GnRH or saline solution intramuscularly. Ultrasonography (US) was performed from D0 to D25. An US approach was used to rank hydrometra in scores. The pregnancy rate was assessed 45 and 90 days after the end of treatment. The uterine fluid was totally drained after the first and second administration of d-cloprostenol in 50% and 95% of the goats, respectively. In one female, full emptying of the uterus occurred only after D20. US performed at 45 and 90 days after the end of treatment indicated there was a pregnancy rate of 45.0% and 55.0%, respectively. Fertility did not differ between the GnRH-treated and control goats. Those goats not pregnant at 45 days had a follicular cyst, hydrosalpinx or hydrometra. At 90 days, no change was observed in the hydrosalpinx, and four goats had hydrometra. The use of three doses of d-cloprostenol 10 days apart was efficient for induction of draining the contents of the uterus, resulting in a relatively acceptable pregnancy rate. This treatment associated with the US approach can be important when applied in the field.

Assessing the usefulness of B-mode and colour Doppler sonography, and measurements of circulating progesterone concentrations for determining ovarian responses in superovulated ewes.

The main goal of this study was to assess the usefulness of two imaging modalities, namely the B-mode and colour Doppler sonography, and serum progesterone (P4 ) concentrations for determining the ovarian response in superovulated ewes. Twenty-four sexually mature Santa Inês ewes underwent the superovulatory treatment consisting of eight injections of porcine FSH (total dose of 200 or 133 or 100 mg; n = 8 ewes/total dose) given at 12-hr intervals and initiated 48 hr before CIDR® (Pfizer Inc., Auckland, New Zealand) removal. Six days after natural mating, the ovaries of all donor ewes were visualized and examined with transrectal ultrasonography and then with videolaparoscopy to identify and enumerate corpora lutea (CL) and luteinized unovulated follicles (LUFs). Jugular blood samples were collected just prior to ovarian examinations. The total number of CL (r = .78 and 0.83, p < .0001) and LUFs (r = .74 and 0.90, p < .0001) enumerated using the B-mode and colour Doppler ultrasonographic technique, respectively, were correlated with that ascertained by videolaparoscopy. Circulating concentrations of P4 were related directly to the number of healthy CL (r = .73, p = .0002) and inversely to the number of prematurely regressing CL (r = -.46, p = .03), but the accuracy of predicting the number of short-lived CL with serum P4 concentrations was very poor. The present results indicate that ultrasonographic imaging and serum P4 measurements on the day of embryo recovery are useful indicators of total/normal CL numbers and both ultrasonographic techniques can be used to quantify LUFs in superovulated ewes.

Colour-Doppler ultrasound imaging as a laparoscopy substitute to count corpora lutea in superovulated sheep.

This study evaluated colour-Doppler ultrasound imaging (UI) as a substitute for laparoscopy to count the corpora lutea (CL) in superovulated sheep. Twenty-five Santa Ines ewes were superovulated three times at 21-day intervals. Corpora lutea were counted by colour-Doppler UI (CLDOPPLER ) 6 days after each superovulation and confirmed by laparoscopy (CLLAP ) 12 hr later. The mean number of CL was similar for both techniques (2.1 ± 2.5 vs. 2.1 ± 2.7 for CLDOPPLER and CLLAP , respectively) with a significant positive correlation (r = .94; r2 =.89). Colour-Doppler UI effectively evaluated the ovarian response in superovulated ewes and efficiently identified animals that did not respond to superovulation.

Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence.

The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 µL-drop) or individually (1 COC/10 µL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 µm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 µm/day (fast-growing) and that reached at least 600 µm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 µm, follicle diameter of at least 600 µm, and oocyte diameter ≥120 µm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.

Transrectal ultrasound evaluation in tropical dairy goats: an indispensable tool for the diagnosis of reproductive disorders.

In Brazil, great milk productivity was achieved after the implementation of a genetic improvement program. However, reproductive efficiency is still far from optimal, possibly due to the high number of undiagnosed disorders that may affect fertility. The aim of this study was to evaluate occurrences of the main reproductive disorders in dairy goats in southeastern Brazil. Data were collected between January 2015 and May 2017 from 23 commercial herds of different breeds, with goats ranging from 8 months to 12 years of age. Transrectal ultrasound exams were performed in 2680 goats. A total of 14.8% of the does showed a disorder in the reproductive tract: hydrometra (10.0%), ovarian follicular cysts (2.3%), gestational loss (1.5%), and hydrosalpinx (1.1%). This was the first study evaluating reproductive disorders in live animals that used a high number of Brazilian dairy goats. Considering that all these diseases affect fertility to different degrees, the performance of transrectal ultrasonography exams twice a year is strongly suggested, in order to guide precocious treatment or discard the animal as soon as possible, thus reducing economic losses in dairy goat farming.

Cervical relaxation for non-surgical uterus access in Santa Inês ewes / Relaxamento cervical para acesso uterino não cirúrgico em ovelhas Santa Inês

The present study was composed by two experiments aiming to develop a cervical dilation technique for non-surgical access to Santa Inês ewe's uterus. In Experiment 1, thirty ewes underwent four epidural treatments. The three experimental treatments used 2.0mg/kg ketamine. The group receiving this drug alone was denominated KG, whereas other group had ketamine associated with 0.1mg/kg morphine (KM) and KX a third group had ketamine associated with 0.05mg/kg xylazine (KX). Control treatment was 1mL/7.5kg saline solution epidurally (CON). Cervical dilation was evaluated in both experiments by attempting to pass a metal rod through the cervix. In Experiment 2, three different hormonal protocols for cervical dilation were tested in thirty ewes. Epidural anesthesia with 2.0mg/kg ketamine was the control treatment (KG) and was combined with hormonal treatments: Misoprostol (MI); Oxytocin + Estradiol (OE); Misoprostol + Oxytocin + Estradiol (MOE). In Experiment 1 transposition rate was not different among groups. In Experiment 2, OE presented the highest rate (90%) while MOE presented 86.2%, MI 68.9% and CON 62.1%. The study developed a pharmacological protocol that increased cervical transposition making the non-surgical access to the uterus feasible in Santa Inês ewes.(AU)

O presente estudo teve como objetivo desenvolver uma técnica para acesso não cirúrgico ao útero de ovelhas Santa Inês e foi realizado em duas etapas. No experimento 1, 30 ovelhas foram submetidas a quatro tratamentos epidurais. Os três tratamentos teste usaram 2,0mg/kg de cetamina. O grupo que recebeu apenas cetamina foi denominado KG, enquanto no segundo grupo a cetamina foi combinada com morfina (0,1mg/kg - KM) e um terceiro grupo recebeu cetamina associada com xilazina 0,05mg/kg - KX). No grupo controle (CON), usou-se solução salina (1mL/7,5kg de peso). A dilatação cervical foi avaliada em ambos os experimentos pela tentativa de transposição cervical com uma haste metálica. No experimento 2, três protocolos hormonais de dilatação cervical foram testados em 30 ovelhas. Anestesia epidural com 2,0mg/kg de cetamina foi o tratamento controle (CON), combinada com tratamentos hormonais nos grupos: misoprostol (MI); ocitocina + estradiol (OE); misoprostol + ocitocina + estradiol (MOE). No experimento 1, a taxa de transposição não variou entre os grupos. No experimento 2, OE teve a maior taxa (90%), enquanto MOE apresentou 86,2%, MIi 68,9% e CON 62,1%. O estudo desenvolveu um protocolo farmacológico que aumentou a taxa de transposição cervical, tornando o acesso uterino não cirúrgico viável em ovelhas Santa Inês.(AU)

Parâmetros reprodutivos após indução de estro com diferentes intervalos de permanência do dispositivo vaginal em cabras Toggenburg acíclicas / Reproductive parameters after induction of estrus with different ranges of the vaginal device in acyclic Toggenburg goats

Este estudo objetivou avaliar o comportamento sexual e a dinâmica ovulatória de cabras da raça Toggenburg após a indução do estro com dispositivo intravaginal de progesterona durante seis, nove e 12 dias de permanência. No momento da inserção do dispositivo e 24 horas antes da retirada do dispositivo, foi administrado 5mg de dinoprost e 200UI de eCG, respectivamente. A dinâmica ovulatória foi acompanhada por ultrassonografia a cada oito horas, enquanto o estro foi observado a cada 12 horas. Todas as cabras apresentaram estro. O intervalo da retirada do dispositivo ao início do estro foi de 29,5 ± 9,6 para G6dias; 34,0 ± 6,0 e 32,4 ± 7,7h, G9dias e G12dias, respectivamente. Igualmente, foi encontrada diferença (P<0,05) entre o G6dias e os outros grupos. A duração do estro diferiu (P<0,05) entre o G6dias (36,0 ± 12,6) e os outros dois grupos (G9dias: 31,2 ± 14,3; G12dias: 33,4 ± 8,6). A taxa de ovulação (81%; 89,5% e; 71,4%), intervalo da retirada do dispositivo à ovulação (50,5 ± 11,4; 46,3 ± 5,9 e; 46,7 ± 8,3), diâmetro do maior folículo (6,6 ± 0,3; 6,6 ± 0,9 e; 6,9 ± 0,8) diâmetro do segundo maior folículo (6,4 ± 0,7; 6,6 ± 0,8 e; 6,8 ± 0,6) e o número de folículos ovulados (1,5 ± 0,6; 1,7 ± 0,6 e 2,0 ± 0,9) para G6dias, G9dias e G12dias, respectivamente, foram semelhantes (P>0,05). Todos os protocolos foram eficientes em induzir o estro sincronizado em cabras da raça Toggenburg, trabalhando em conjunto com a eficiência reprodutiva e produtiva desses animais.(AU)

This study aimed to evaluate the sexual behavior and the ovulatory dynamics of Toggenburg goats after induction of synchronized estrus by an intravaginal progesterone device for six, nine and twelve days. At the device insertion and 24 h before the device removal, 5mg of dinoprost and 200 UI of eCG was administered, respectively. The ovulatory dynamics was assessed by ultrasound every 8 h, while the sexual behavior was observed every 12 h. All goats showed estrus. The intervals from device removal to estrus were 29.5 ± 9.7, 34.0 ± 6.0 and 32.4 ± 7.7h to G6days, G9days and G12days, respectively. Iqually, it was found difference (P<0.05) among G6days and the other groups. The duration of estrus differed (P<0.05) among the G6days (36.0 ± 12.6) and the other two groups (G9days: 31.2 ± 14.3; G12days: 33.4 ± 8.6). The ovulation rate (81.0%; 89.5% and; 71.4%); the interval from device removal to ovulation (50.5 ± 11.4; 46.3 ± 5.9 and; 46.7 ± 8.3), the diameter of the largest follicle (6.6 ± 0.3; 6.6 ± 0.9 and; 6.9 ± 0.8), diameter of the second largest follicle (6.4 ± 0.7; 6.6 ± 0.8 and; 6.8 ± 0.6) and the number of ovulations (1.5 ± 0.6; 1.7 ± 0.6 and; 2.0 ± 0.9) for G6days, G9days and G12days, respectively, were similar (P>0.05). All treatments were effective for the induction of synchronized estrus in Toggenburg goats, working jointly with the reproductive and productive efficiency of these animals.(AU)

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Effect of increasing amounts of milk replacer powder added to whole milk on postweaning performance, reproduction, glucose metabolism, and mammary fat pad in dairy heifers.

The aim of this study was to evaluate the effects of increasing the total solids (TS) content of liquid feed, by adding increasing amounts of milk replacer powder to whole milk, on age at puberty and postweaning performance, glucose metabolism, and mammary fat pad of dairy heifers. Crossbreed Holstein × Gyr heifers (n = 58) were distributed into 4 different treatments during the preweaning period. Treatments consisted of liquid feeds with TS content of 12.5 (actual TS = 13.5 ± 0.53%; n = 15), 15.0 (actual TS = 16.1 ± 0.03%; n = 15), 17.5 (actual TS = 18.2 ± 0.14%; n = 13), or 20.0% (actual TS = 20.4 ± 0.24%; n = 15), which were provided up to 59 d old. From 60 to 89 d old, the animals received the same starter offered during the preweaning period in addition to corn silage ad libitum starting at 70 d old. From 90 to 104 d old, the animals were adapted to a total mixed ration. At 105 d old, the animals were distributed in 4 paddocks equipped with electronic feed and water bins and were fed the same total mixed ration ad libitum. A period of adaptation to the electronic feed bins occurred from 105 to 119 d old. Feed and water intake and body weight and body frame development were assessed until puberty. Mammary gland evaluations were performed monthly by ultrasonography from 120 d of age until puberty onset. Puberty onset was determined as plasma progesterone concentration greater than 1 ng/mL in 2 consecutive samples collected 7 d apart. The date of puberty onset was recorded as the collection day of the first of these samples. A glucose tolerance test was performed at 280 d of age. The increased TS content of the liquid feed fed during the preweaning period did not affect dry matter intake, performance, age at puberty, glucose metabolism, or mammary gland fat pad at later stages of rearing process. Conversely, hip width and heart girth increased linearly up to 150 and 240 d of age, respectively, as a result of the increased TS concentration of the liquid feed during the preweaning period.

Serum testosterone, sperm quality, cytological, physicochemical and biochemical characteristics of the prostatic fraction of dogs with prostatomegaly.

Prostatomegaly is a common finding in older non-neutered dogs. This study compared the serum testosterone, sperm quality and characteristics of the prostatic fraction between healthy dogs and dogs with prostatomegaly. Blood samples of ten dogs (five dogs from each group) were taken for serum testosterone measurement. Sperm motility, vigour, concentration, viability, membrane functionality and morphology were analysed in sperm-rich fraction. Osmolality, pH, cell types, and albumin, haemoglobin, acid phosphatase, alkaline phosphatase, glucose, triglycerides, cholesterol, calcium, phosphorus, magnesium and chloride were analysed in prostatic fraction. Dogs with prostatomegaly have the lowest sperm motility, vigour, concentration and functional membrane. Dogs with prostatomegaly have the highest glucose, triglycerides and cholesterol. Glucose was the only constituent positively correlated with serum testosterone and prostate volume. It can be concluded that dogs with prostatomegaly have poorer sperm quality, and glucose, triglycerides and cholesterol in prostatic fraction can be used as prostatomegaly biomarkers.

Serum profile of cytokines interferon gamma and interleukin-10 in ewes subjected to artificial insemination by cervical retraction.

This study evaluated the influence of artificial insemination (AI) by cervical retraction (CRI) on serum levels of interferon gamma (IFNγ) and interleukin-10 (IL-10) in ewes. Synchronized pluriparous Santa Inês ewes were subjected to natural mating (NM, n = 8) and AI, which was performed for a fixed time (55 ± 1 hour) by CRI (n = 8) or laparoscopy (n = 8). Ewes were classified as pregnant, with return to estrus (RE) or with embryonic loss (EL). Blood samples were collected on Day 0, Day 3, Day 5, Day 12, and Day 17 (Day 0 = AI/NM) for progesterone dosage and cytokines were quantified from Day 0 to Day 12. Progesterone levels were constant, except for a decrease in ewes with RE at Day 17 (P < 0.05). Regardless of the reproductive method used, there was no difference in the IFNγ and IL-10 levels at any time, with averages of 642.1, 713.2, and 741.2 pg/mL for IFNγ and 667.1, 616.8, and 721.1 pg/mL for IL-10 when using CRI, laproscopy, and NM, respectively. Regarding the physiological status, ewes with EL had lower serum levels of IFNγ and IL-10 than pregnant ewes and ewes with RE, regardless of the reproductive method used, with averages of 769.1, 714.9, and 555.7 pg/mL for IFNγ and 713.8, 699.3, and 578.7 pg/mL for IL-10 in pregnant ewes, ewes with RE and EL, respectively (P < 0.01). In conclusion, AI by CRI in Santa Inês ewes does not alter the profile of serum cytokines IFNγ and IL-10 and does not induce an inflammatory reaction that can compromise pregnancy.