Ricarda Maria dos Santos

Assessment of in vitro sperm characteristics and their importance in the prediction of conception rate in a bovine timed-AI program

The aims of this study were to assess in vivo fertility and in vitro sperm characteristics of different sires and to identify sperm variables important for the prediction of conception rate. Multiparous Nelore cows (n = 191) from a commercial farm underwent the same timed artificial insemination (timed-AI) protocol. Three batches of frozen semen from three Angus bulls were used (n = 9). A routine semen thawing protocol was performed in the laboratory to mimic field conditions. The following in vitro sperm analyses were performed: Computer Assisted Semen Analysis (CASA), Thermal Resistance Test (TRT), Hyposmotic Swelling Test (HOST), assessment of plasma and acrosomal membrane integrity, assessment of sperm plasma membrane stability and of lipid peroxidation by flow cytometry and assessment of sperm morphometry and chromatin structure by Toluidine Blue staining. For statistical analyses, Partial Least Squares (PLS) regression was used to explore the importance of various sperm variables in the prediction of conception rate. The following in vitro sperm variables were determined to be important predictors of conception rate: total motility (TM), progressive motility (PM), TM after 2 h of thermal incubation (TM_2 h), PM after 2 h of thermal incubation (PM_2 h), Beat Cross Frequency after 2 h of thermal incubation (BCF_2 h), percentage of rapidly moving cells after 2 h of thermal incubation (RAP_2 h), intact plasma membrane evaluated by HOST, intact plasma and acrosomal membranes evaluated by flow cytometry, intact plasma membrane suffering lipid peroxidation, major defects, total defects, morphometric width/length ratio, Fourier_0 and Fourier_2 and Chromatin Heterogeneity. We concluded that PLS regression is a suitable statistical method to identify in vitro sperm characteristics that have an important relationship with in vivo bull fertility.

Elevated progesterone concentrations enhance prostaglandin F2α synthesis in dairy cows.

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha); PGFM) following treatment with estradiol-17beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n=9), received 20mg LH (n=7), or had ovarian follicles larger than 6mm aspirated (n=8). In Experiment 2, cows on Day 6 were untreated (n=9) or received 5000 IU hCG (n=10). In Experiments 1 and 2, all cows received 3mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n=4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n=7) received the alternate sequence. Blood was collected daily for measurement of P(4) 4h after concentrate feeding. On Day 17, blood was collected from 1h before to 1h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release of PGF(2alpha) were similar between LH-treated and control cows (P>0.10). In Experiment 2, plasma P(4) was elevated to a greater extent on Day 17 in cows treated with hCG (P<0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P<0.05). In Experiment 3, there was a group x period interaction (P<0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P<0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2alpha) in response to E(2) or OT.

Ovarian activity and oocyte quality associated with the biochemical profile of serum and follicular fluid from Girolando dairy cows postpartum

This study was designed to evaluate the influence of heat stress (HS) on the metabolic profile of serum and follicular fluid (FF), ovarian follicle development, and oocyte quality of Girolando dairy cows. Oocytes, blood, and FF (follicles ≥9mm) samples were obtained at 30, 45, 60, 75, and 90 days postpartum in the summer and winter seasons. During transvaginal follicular aspiration, rectal temperature (RT), body condition score (BCS), number of ovarian follicles, and quality of oocytes were recorded. The ambient air temperature (AT) and relative humidity (RH) were also recorded to calculate the temperature humidity index (THI). Glucose, total cholesterol (TC), triglycerides (TG), urea, sodium (Na), potassium (K), and calcium (Ca) concentrations were determined using serum and FF samples. The RT, THI, and BCS loss were greater (P<0.01) in the summer; however, glucose, Na, and K serum concentrations decreased in the same season (P<0.05). Degenerated oocytes were positively associated (P<0.05) with THI (r=0.14) and AT (r=0.13), and negatively associated with glucose (r=-0.12) and K (r=-0.11) serum concentrations. HS induces metabolic changes, which compromise the number of ovarian follicles and the follicular environment, thus resulting in morphologically damaged oocytes.

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.

Inseminação artificial em tempo fixo e diagnóstico precoce de gestação em vacas leiteiras mestiças

It was evaluated, during a period of one year, the reproductive performance of 94 Bos taurus x Bos indicus crossbred dairy cows submitted to an assisted reproduction program. A timed artificial insemination (TAI) protocol was carried out by using an intra-vaginal progesterone device containing progesterone and through injections with Prostaglandin F2α and estradiol cypionate. By using ultrasound, from 7 to 14 days after inseminations or controlled mattings, detection of corpus luteum in the ovaries was done to determine ovulation rate and, on the 28th day, diagnosis of pregnancy was carried out for calculation of conception rate. Treatment only started after a 34-day post-partum minimal period. Body condition score, presence of corpus luteum in the beginning of the protocol, reutilization of intra-vaginal device and controlled mating or artificial insemination did not influence rates of ovulation, conception and conception of ovulated cows. Rates of conception and rates of conception of ovulated cows were negatively affected by the higher number of days post partum (DPP), or days in milk and by the warmer seasons of the year, spring/summer. Response to protocol of timed artificial insemination based on the use of progesterone, PGF2α and estradiol cypionate is negatively affected by lactation days and by the warm season of the year. Body condition does not affect the response to protocol of artificial insemination provided that treated cows show body condition scores higher than 2.5 points.

Metabolic profile of serum and follicular fluid from postpartum dairy cows during summer and winter.

This study was designed to monitor the biochemical profiles of serum and follicular fluid (FF) of postpartum dairy cows during the summer (n=30) and winter (n=30). Blood and FF (follicles ≥ 9 mm) were obtained from Girolando cows at 30, 45, 60, 75 and 90 days postpartum. The samples were collected and analysed to determine glucose, total cholesterol (TC), triglyceride (TG), urea, sodium (Na), potassium (K) and calcium (Ca) levels. Throughout the study, the following clinical variables were measured: rectal temperature (RT), respiratory rate (RR) and body condition score (BCS). In addition, the temperature humidity index (THI) was calculated for each season. During the summer season, THI was higher, BCS decreased, there was an increase in RT, and glucose, urea, Na and K serum levels were decreased (P<0.05). The levels of TC, TG, urea, K and Ca in follicular fluid increased (P<0.05). Positive correlations (P<0.05) were observed between the serum and FF levels for glucose (r=0.29), TC (r=0.24) and Ca (r=0.30). Therefore, the biochemical profile of serum and FF of dairy cows under summer heat-stress conditions demonstrates marked changes that may impair fertility during lactation.

0122 Retained placenta and subclinical endometritis: Prevalence and relation with reproductive performance in crossbred dairy cows.

This study aimed to evaluate the effects of type of calving (eutocic vs. dystocic), season of the year (rainy vs. dry) on retained placenta (RP) and subclinical endometritis (SE) prevalence, to verify the relation between these diseases, as well as its effects on culling rate, days open and number of AI/conception. Retention of fetal membranes was recorded on first day postpartum. Endometrial cytology was performed between 30 and 80 days in milk (DIM) and positive cases were considered ≥ 5% neutrophils. Data were analysed by logistic regression and analysis of variance on Minitab program (P 0.05). In conclusion, RP tended to be a risk factor for SE, dystocia is a predisposing factor for RP and calvings that happens during rainy period increases SE. There is a negative impact on reproductive efficiency of crossbred dairy cows that had retained placenta.

Perfil metabólico de vacas mestiças leiteiras com baixo escore de condição corporal no periparto

ABSTRACT.- Oliveira R.S.B.R., Moura A.R.F., Padua M.F.S., Barbon I.M., Silva M.E.M., Santos R.M., Mundim A.V. & Saut J.P.E. 2014. [Metabolic profile in crossbred dairy cows with low body condition score in the peripartum period.] Perfil metabolico de vacas mesti-cas leiteiras com baixo escore de condicao corporal no periparto. Pesquisa Veterinaria Bra-sileira 34(4):362-368. Faculdade de Medicina Veterinaria, Universidade Federal de Uber-lândia, Uberlândia, MG 38400-902, Brazil. E-mail: jpsaut@famev.ufu.brThe aim of this study was to evaluate the metabolic profile of protein, energy and en-zyme in crossbred dairy cows with low body condition score (BCS) in the peripartum peri-od. Blood samples were collected from 36 animals with 2.6±0.5 BCS, eutocia, physiological postpartum and without any treatment, on following days: one week before calving, calving and 7, 14, 21, 28 and 43 days in milk (DIM). It was evaluated serum total protein, albumin and globulins for protein profile; AST, ALT, GGT and alkaline phosphatase for mineral pro-file; nonesterified fatty acids (NEFA), b-hydroxybutyrate (BHBA), triglycerides, cholesterol and lipoproteins (VLDL, HDL and LDL) for energy profile. Crossbred dairy cows had hy-poproteinemia, hypoalbuminemia, hypocholesterolemia and increased that both enzymes AST and GGT at the precalving. There were lipolysis and hypoglobulinemia at parturition. It was concluded that crossbred dairy cows with low BCS have negative energy balance, hypoproteinemia with hypoalbuminemia and hepatic injury in the peripartum. This con-dition is restored at 30 DIM, but there is no recovery of the body condition by the end of puerperium.INDEX TERMS: Dairy cattle, biochemistry profile, metabolic profile, postpartum, calving, eutocia.

Cisto ovariano em vacas de leite: incidência, resposta à aplicação de GnRH e desempenho reprodutivo

The incidence of ovarian cysts, response to GnRH treatment, and effects on reproductive performance and culling rate of Holstein cows were determined. Ovaries of lactating cows (n=333), were weekly monitored by ultrasound, beginning at fourth week postpartum, to determine the presence of corpus luteum (CL) and follicles greater than 10mm. In the seventh week the cows were classified as cycling (n=248; presence of corpus luteum (CL) in one of the ultrasound evaluations); anovulatory (n=54; absence of CL and follicles less than 25mm), and cystic (n=31; absence of CL and presence of structures greater than 25mm). The cysts cows were distributed in two groups in the seventh week: control group (n=16; without treatment) and treatment group (n=15; cows received one GnRH injection). The recovery rate was 60.0% in treated cows and 87.5% in control cows. The cystic cows had longer average interval from parturition to first AI (P<0.05; 91.4±8.3 vs. 77.8±2.5), higher number of services per conception (P<0.05; 4.4±1.2 vs. 3.6±1.5), longer interval parturition to conception (P<0.05; 214.8± 25.9 vs. 174.9±7.7), and higher percentage of culled cows (P<0.05; 41.2% vs. 21.8%), than cycling cows. Cystic cows without treatment had the same recovery rate as cows treated with GnRH. Cystic cows had longer intervals from parturition to conception, and higher culling rate.

Eficácia da dose reduzida de gonadorelina e diferentes prostaglandinas no protocolo ovsynch em vacas holandesas

The efficacy of 150mcg D-cloprostenol or 0.530mg cloprostenol sodium on the 7- day corpus luteum (CL) regression rate, and of two doses of gonadorelin, 0.10mg vs. 0.25mg, on the ovulation and synchronization rates during the Ovsynch protocol were evaluated. Lactating Holstein cows (n=136), producing 23.75.8kg milk/d and 138.4±72.0 days in milk were randomly assigned to four groups, according to dose of gonadorelin and prostaglandin type. No effect of the products and doses tested on conception rate were observed. The ovulation and synchronization rates using 0.10mg or 0.25mg of gonadorelin were 52.9% and 80.9% vs. 57.4% and 80.9%, respectively. The CL regression rates in cows ovulating after the first GnRH using D-cloprostenol or cloprostenol sodium were 97.1% and 97.5%, respectively. The pregnancy rate was not affected by treatment, but by ovulation rate after the first injection of GnRH. The pregnancy rates were 16.0% and 6.6% for ovulated and non ovulated cows, respectively. The results suggest that 0.10mg dose of gonadorelin was efficient and both prostaglandins can be used in synchronization of ovulation protocols.