Mara Iolanda Batistella Rubin

Calves born after open pulled straw vitrification of immature bovine oocytes.

The aim of this study was to evaluate the developmental capacity of immature bovine oocytes after vitrification with 20% ethylene glycol (EG)+20% dimethyl sulfoxide (Me(2)SO) and 0.5M sucrose (SUC), by open pulled straw (OPS) technology. The effect of treatment with cytochalasin D before vitrification was also examined. No differences were observed in cleavage and blastocyst rates among the group vitrified without cytochalasin D treatment (Vitri) (49.0% and 6.1%) and that with cytochalasin D treatment before vitrification (CDVitri) (46.4% and 3.6%), but both were lower (P<0.05) than the unvitrified control group (85.1 and 45.9%). Calves were obtained after transfer of fresh and vitrified blastocysts from the Vitri group and after transfer of vitrified blastocysts from the CDVitri group. Cytochalasin D treatment does not improve the development of immature bovine vitrified oocytes. The results show that a small proportion of immature oocytes vitrified with this technology are fully competent to produce blastocysts, which may be transferred immediately or vitrified before transfer, and go on to develop healthy offspring.

Effect of the time of artificial insemination with frozen-thawed or fresh semen on embryo viability and early pregnancy rate in gilts

The aim of the present study was to evaluate the effect of artificial insemination time (before or after ovulation) using either fresh or frozen-thawed boar semen on embryo viability and early pregnancy rate. Seventy-seven prepubertal crossbred (Landrace x Large White x Duroc) gilts were inseminated in 4 treatments. Artificial inseminations were performed 6 h either after (A) or before (B) ovulation using frozenthawed (A-frozen, n = 19; B-frozen, n = 19) or fresh semen (A-fresh, n = 21; B-fresh, n = 18). The gilts were induced to puberty by administration of 400 IU of eCG and 200 IU hCG (sc) followed by 500 IU of hCG (sc) 72 h later. Ovulation was predicted to occur 42 h after the second injection. All animals were slaughtered 96 h after AI. Embryos were collected and classified as viable (5- to 8-cells, morulae, compacted morulae and early blastocysts) and nonviable (fragmented, degenerated and 1- to 4-cell embryos). The total embryo viability rate was: 64.3% (A-frozen), 54.2% (A-fresh), 76.0% (B-frozen), 91.9% (B-fresh); (A-fresh vs B-fresh, P = 0.018; A-frozen vs B-frozen, P = 0.094). It was observed that AI before ovulation resulted in a higher percentage of total viable embryos than AI after ovulation (P = 0.041). The early pregnancy rate, defined as presence of at least one viable embryo, was 78.9, 80.9, 84.2 and 94.4% for A-frozen, A-fresh, B-frozen, B-fresh, respectively. There was no significant difference in the early pregnancy rate among groups. In conclusion, there was a detrimental effect upon total embryo viability rate when AI was performed after ovulation with either frozen-thawed or fresh semen. The total embryo viability rate and the early pregancy rate were not affected by AI with either frozen-thawed or fresh semen regardless of the time of AI.

Prostaglandin F2α promotes ovulation in prepubertal heifers

The objective was to determine the effects of exogenous prostaglandin F(2α) (PGF), with or without progesterone treatment, on first ovulation in prepubertal heifers. We tested the hypothesis that PGF has a luteolysis-independent ovulatory effect in cattle. Crossbred Angus heifers (12 to 14 mo old, 250 kg body weight, and an average body condition score of 3 out of 5) were examined by transrectal ultrasonography on two occasions, 11 days apart. Heifers in which a CL was not detected at either examination were considered prepubertal. Heifers were assigned randomly to three experimental groups: (1) PG group (N = 14); heifers were treated with a PGF analog (500 μg cloprostenol im) 5 days after the emergence of a spontaneous (i.e., naturally occurring, noninduced) follicular wave; (2) PPG group (N = 12); heifers were given an intravaginal progesterone-releasing insert (CIDR; Pfizer Animal Health, Montreal, QC, Canada), and a follicular wave was induced with 50 mg of progesterone + 2 mg of estradiol benzoate im, and a PGF analog was given at the time of CIDR removal, on day 5 of the follicular wave (on average, 8.6 ± 0.5 days after CIDR insertion); and (3) control group heifers were given no treatment (N = 14). Heifers were examined daily by transrectal ultrasonography from the start of the experiment to confirmation that ovulation had occurred, or to 5 days after PGF injection (PG and PPG groups) or until dominant follicles of the next follicular wave reached 8 mm (control group). The percentage of heifers that ovulated within 10 days after wave emergence was higher in PPG (10/12; 83.3%) and PG (11/14; 78.5%) groups than in control (1/14; 7.1%; P < 0.0001). Ovulations occurred 69.6 ± 6 h and 93.8 ± 5 h after PGF treatment in PPG and in PG groups, respectively, whereas only one heifer in the control group ovulated 96 h after day 5 of follicular wave (P = 0.13). In summary, PGF treatment was associated with ovulation in prepubertal heifers whether or not exogenous progesterone was used as a pretreatment. The hypothesis that PGF will induce ovulation by a luteolysis-independent mechanism was supported.

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.

Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol

This study evaluated the effect of different doses of eCG (control, 300 or 400 IU) administered at progesterone (P4) device removal in suckled Bos taurus beef cows undergoing a timed artificial insemination (TAI) protocol. A total of 966 cows received a P4 insert and 2.0 mg intramuscular estradiol benzoate at the onset of the synchronization. After 9 days, P4 insert was removed, and 12.5 mg of dinoprost tromethamine and 1 mg of estradiol cypionate were administered, followed by TAI 48 hours later. Then, the cows received one of three treatments as follows: control (n = 323), 300 (n = 326), or 400 IU of eCG (n = 317). A subset (n = 435) of cows in anestrus had their ovaries evaluated using ultrasound at the time of P4 removal and at TAI. Data were analyzed by orthogonal contrasts (C): C1 (eCG effect) and C2 (eCG dose effect). Estrous occurrence (control = 53.7%, 300 IU = 70.6%, and 400 IU = 77.0%) and pregnancy per artificial insemination (control = 29.7%, 300 IU = 44.8%, and 400 IU = 47.6%) were improved by eCG treatment (C1; P = 0.0004 and P < 0.0001, respectively). Furthermore, the cows receiving eCG presented larger follicles at TAI (control = 13.5 ± 0.3 mm, 300 IU = 14.0 ± 0.2 mm, and 400 IU = 15.1 ± 0.3 mm; P < 0.0001; C1). However, there was no effect of eCG dose on any response variables studied (C2; P > 0.15). In conclusion, the eCG treatment administered at the time of P4 removal increased the occurrence of estrus, the larger follicles at TAI, and pregnancy per artificial insemination of suckled B taurus beef cows. Despite the greater occurrence of estrus in noncyclic cows receiving 400 IU of eCG, both eCG doses (300 and 400 IU) were equally efficient to improve pregnancy to artificial insemination.

TÉCNICAS DE COLORAÇÃO CROMOSSÔMICA PARA ESTÁGIOS ESPECÍFICOS DA MATURAÇÃO NUCLEAR DE OÓCITOS BOVINOS

This study was designed to determine the efficiency of three different technics for staining bovine oocyte in the germinal vesicle (GV), metafase I (MI) and metafase II (MII) stages. In these stages, oocytes without cumulus cells were fixed in acetic acid:methanol (1:3) either on slides (FLL) or in petri dishes (FPL) and stained with 1% of lacmoid in phosphate buffered saline (PBS). Also, the oocytes were randomized divided in a third treatment, in which they were fixed on slides immersed in acetic acid:methanol (1:3) and stained with Giemsa (FLG). In the GV stage, the percentage of oocytes accurately identified in the FLL (93.5%) was significantly higher than in the FPL (53.1%; χ2=9.84; p=0.0017) and FLG (55.2%; χ2=9.03; p=0.0027) treatment groups. However, the proportion of oocytes in the MI and MII stages correctly stained with FLL technic (MI 41.7% and MII 41.9%) was statistically lower than that observed in the FPL (MI 90.0%, χ2=13.25; p=0.0003, and MII 92.8%, χ2=12.45; p=0.0004) and FLG (MI 83.3%, χ2=10.69; p=0.0011, and MII 89.7%, χ2=12.24; p=0.0005) treatment groups. With these results, it can be established that the efficiency of the technic to fix and to stain bovine oocyte depends on the nuclear maturation stage to be investigated.

The use of PGF2α as ovulatory stimulus for timed artificial insemination in cattle

The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In experiment 1, crossbred dual-purpose heifers, in a crossover design (3 × 3), were given an intravaginal progesterone-releasing insert (controlled internal drug release [CIDR]) plus 1 mg estradiol benzoate (EB) intramuscularly (im) and 250 μg of a PGF-analogue im on Day 0. The CIDR inserts were removed 5 days after follicular wave emergence, and the heifers were randomly divided into three treatment groups to receive the following treatments: (1) 1 mg of EB im (EB group, n = 13); (2) 500 μg of PGF im (PG group, n = 13); or (3) saline (control group, n = 13), 24 hours after CIDR removal. Ovulation occurred earlier in EB (69.81 ± 3.23 hours) and PG groups (73.09 ± 3.23 hours) compared with control (83.07 ± 4.6 hours; P = 0.01) after CIDR removal. In experiment 2, pubertal beef heifers (n = 444), 12 to 14 months of age were used. On Day 0, the heifers were given a CIDR insert plus 2 mg EB im. On Day 9, the CIDR was removed and the heifers were given 500 μg of PGF im. Heifers were randomly assigned into one of three treatment groups: (1) 1 mg of EB (EB group; n = 145); (2) 500 μg of PGF (PG group; n = 149), both 24 hours after CIDR removal; or (3) 600 μg of estradiol cypionate (ECP group; n = 150) at CIDR removal. Timed artificial insemination occurred 48 hours after CIDR removal in the ECP group and 54 hours in the PG and EB groups. The percentage of heifers ovulating was higher in the PG group compared with the other groups (P = 0.08). However, the pregnancy rates did not differ among groups (47.6%, 45%, and 46.6%, for EB, PG, and ECP, respectively; P = 0.9). In experiment 3, 224 lactating beef cows, 40 to 50 days postpartum with 2.5 to 3.5 of body condition score were treated similarly as described in experiment 2, except for the ECP group, which was excluded. The treatments were as follows: 1 mg EB (EB group; n = 117) or 500 μg PGF (PG group; n = 107), 24 hours after CIDR removal. The calves were temporarily separated from their dams from Days 9 to 11. No difference was detected on the pregnancy rate between the EB and PG groups (58.1% vs. 47.6%, respectively; P = 0.11). Taken together, the combined results suggested that PGF2α could be successfully used to induce and synchronize ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with other ovulatory stimuli (ECP and EB).

Desenvolvimento embrionário in vitro de oócitos bovinos mantidos em líquido folicular ou TCM-hepes

In order to evaluate the effect of a transport medium on the rate of in vitro embryonic development, 1381 Cumulus-oocyte Complexes (COC) were obtained by aspiration of 2-8mm diameter follicles witch were randomly divided in 4 treatment groups. The Control group was formed by oocytes matured in modified TCM-199 for 24h, incubated at 39°C and 5,00% CO2 with saturated humidity. The group 1 (WB24h), included oocytes matured in 1.0mL tubes containing TCM-HEPES (5.95mg/mL), in water bath (WB) at 39°C for 24h. The group 2 (FFb6C18h), included oocytes kept in bovine follicular fluid (FFb) for 6h at 30°C followed by a period of 18h maturation under the same conditions as the Control group and with the oocytes maintained in FFb followed by 18h IVM under the same conditions as the group 1, group 3 (FFb6WB18h). Fertilization was performed in FERT-TALP for 18h. Zygotes were cultured in SOFaaci under mineral oil within gasified bags. The cleavage rate differed (P<0.05) between the Control and FFb6BM18h groups. However, there was no difference on the D7 and D9 blastocyst rates and on the percentage of blastocyst ecloded. It was concluded that it is possible to maintain the oocytes in FFb for 6h at 30°C before 18h IVM, or to proote the transport and maturation of the oocytes for 24h, in TCM-HEPES and water-bath at 39°C, without compromising embryonic development. The simplification of MIV showed in this experiment through of tubes (1.0mL) replete with TCM-HEPES and holding in water bath at 39°C, could be a viable and practice for the bovine programs of OPU/PIV.

Qualidade do leite em amostras individuais e de tanque de vacas leiteiras

Bovine milk samples were collected from milk tanks (n = 69) and from individual cows (n = 3,517) on specialized (S, n = 3), partially specialized (PS, n = 5) and nonspecialized (NS, n = 7) production systems. Compositions, somatic cell count (SCC) and urea nitrogen content were analyzed for the different production systems and all the four seasons. Data were compared to the ranges allowed by the Federal Normative Instruction 51/2002 (IN51) for South Brazil from May 2009 to June 2010. Forty-two percent (n = 29/69) of all milk samples obtained from tanks and 11% of the individual samples in the three production systems met IN51 standards. Of the collected tank samples, 70% (n = 14/20), 39% (n = 9/23) and 23% (n = 6/26) of the S, PS and NS systems, respectively, met IN51 criteria. These data indicate that evaluation of individual samples yields a lower percent of conformity with IN51 standards than tank samples. This implies that the milk from healthy cows may dilute the higher SCC of diseased cows, suggesting that tank samples are not accurate for a confident milk quality indicator. The fat, protein and total solids content in the tank samples were similar among the three production systems. Average lactose and urea nitrogen content in tank samples were similar between seasons. In contrast, the average SCC was above the IN51 standard in the tank and individual samples during the fall. Urea nitrogen content average was highest (P < 0.001) in the specialized production system, both in the tank and individual samples. The evaluation of individual samples may improve nutritional support of the specialized system.

Cultivo individual de blastocistos bovinos produzidos in vitro

In vitro produced bovine embryos were individually cultured from D7 to D9 to observe their development. Forty-nine blastocysts (early blastocyst, blastocyst and expanded blastocyst) were individually cultured, from D7 to D9 (D0= fertilization), in 50ml SOF medium plus 5% OCS. Between D7 and D8, blastocysts were cultured in straws (SC) or in plates (PC) and from D8 up to D9, they were cultured on plates. The ratio of early blastocyst, blastocyst, and expanded blastocyst advancing at least one stage of development were, respectively, 71%, 37%, 44% in PC and 100%, 66%, 36% in SC (P>;0.05). Overall development rates on D8 were not significantly different (P>;0.05) for PC (50%) and SC (60%). At D9, both culture systems produced similar (P>;0.05) hatching rates (29% and 24% for PC and SC, respectively). After fluorescent nuclei staining, the average number of cells counted in the hatched (182.7 vs. 202.8) and expanding (94.5 vs. 88.0) blastocysts were similar (P>;0.05) for PC and SC, respectively. In vitro produced bovine blastocysts can be individually cultured from D7 to D9, and the culture system employed (dishes or straws) has no effect on hatching rates and cell number of developed blastocysts.