Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos

Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle

Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30–50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15–23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14–18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves (≈20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal

Early pregnancy diagnosis by palpation per rectum: influence on embryo/fetal viability in dairy cattle

The objective was to estimate the effect of palpation per rectum (for early pregnancy diagnosis) on embryo/fetal viability in dairy cattle. A controlled, randomized block-design experiment with two blocks, one by category, and the other by number of embryos, was conducted. Five-hundred-and-twenty pregnant dairy cows and heifers with a viable embryo detected by transrectal ultrasonography (TRUS) between days 29 and 32 after AI were included. The pregnant females were randomly allocated into two nearly equal groups: palpation per rectum (PAL group; n=258) and no palpation per rectum (NPAL group; n=262). The PAL group was submitted to palpation per rectum (PPR) using the fetal membrane slip (FMS) technique once between days 34 and 41 of pregnancy. The fetal membrane slip consisted of compressing the pregnant uterine horn and allowing the chorioallantoic membrane to slip between the fingers. Both groups were submitted to two additional TRUS at days 45 and 60 of pregnancy, to monitor the potential immediate and delayed deleterious effects of PPR on embryo and fetal viability, respectively. A diagnosis of embryo/fetal death was made when there was no embryo/fetal heart beat or the absence of positive signs of pregnancy in an animal previously diagnosed pregnant, or the presence of signs of embryo/fetal degeneration. The overall rate of embryo/fetal death was 14.0% (73/520). Embryonic death (10%; 52/520) was higher than fetal death (4.5%; 21/468; P\u3c0.001). Embryo/fetal mortality was higher in cows (16.4%; 59/360) than in heifers (8.8%; 14/160; P\u3c0.025) and in cattle with twin (25.5%; 12/47) versus singleton pregnancies (12.9%; 61/473; P\u3c0.025), but was not different (P\u3e0.05) between PAL (14.7%; 38/258) and NPAL (13.4%; 35/262). In conclusion, PPR between days 34 and 41 of pregnancy using the fetal membrane slip technique did not affect embryo/fetal viability

Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle

The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P\u3c0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P\u3c0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P\u3c0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P\u3e0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P\u3c0.05) and the specificity was 93.0 and 93.4% (P\u3e0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively)

Effects of early pregnancy diagnosis by palpation per rectum on pregnancy loss in dairy cattle

OBJECTIVE: To determine the effect of palpation per rectum (PPR) by use of 1 or 2 fetal membrane slips (FMSs) for pregnancy diagnosis during early gestation on pregnancy loss in dairy cattle. DESIGN: Controlled, randomized block design. ANIMALS: 928 healthy pregnant cattle. PROCEDURES: All cattle were determined to be pregnant by use of transrectal ultrasonography at approximately day 31 after estrus and randomly allocated into 2 groups (control group [n = 476 cows] and palpation group [452]). The control group was not subjected to pregnancy diagnosis via PPR. The palpation group was subdivided into 2 groups (PPR FMS 1 [n = 230 cows] and PPR FMS 2 [222]), which involved PPR and pregnancy diagnosis via 1 or 2 FMSs, respectively, during the same examination, which was performed by 1 veterinarian between days 34 and 43 after estrus. All cattle were reevaluated by use of transrectal ultrasonography on days 45 and 60 to determine viability of the embryo and fetus, respectively. RESULTS: Overall pregnancy loss between days 31 and 60 was 14.1%. Pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups from days 31 to 60 was 14.5%, 12.6%, and 14.9%, respectively. Embryonic pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups was 12.4%, 9.1%, and 9.5%, respectively. Fetal pregnancy loss for the same groups was 2.4%, 3.8%, and 5.9%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Pregnancy diagnosis via 1 or 2 FMSs performed during PPR in early gestation did not increase pregnancy loss in dairy cattle