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

In Vitro Fertilization of Immature Oocytes by Testicular Sperm: Animal Model for Azoospermic Infertile Patients

Background: Men with azoospermia require testicular and epididymal sperm aspiration for intracytoplasmic sperm injection. Women over 37 years of age demonstrate an increase number of immature oocytes after induction of ovulation. The development of a technique for in vitro maturation (IVM) and fertilization (IVF) of the oocytes using testicular, epididymal and vassal sperm (TS, ES, VS, respectively) will be of therapeutic value for the treatment of azoospermic patients. Objectives: The goal of the study was to develop an animal model for the treatment of infertile couples with obstructive azoospermia and immature oocytes. Materials and Methods: Canine ovaries and testes were collected from a local animal clinic. Immature oocytes were retrieved from the ovaries and cultured in modified tissue culture medium (MTCM). The mature oocytes were in vitro fertilized by TS, ES, and VS. The normality of the fertilized oocytes was studied. Results: Sperm motility index was significantly higher (P<0.01) in sperm retrieved from the vas deference compared to ES and TS. The concentration of the sperm was significantly higher (P<0.05) in the testes compared to epididymis and vas deference while VS and ES normal morphology were significantly (p<0.01) better than TS. Viable oocyte percentage was significantly higher (P<O.05) in 72 hours versus 96 hours culture durations. Significant higher IVF was reported in VS compared to other groups (P < 0.05) and the SPI was significantly higher after 96 versus 72 hours duration (P<O.05). Conclusion: IVM of the oocytes in MTCM for 72 and 96 h resulted in a significant increase in IVM rate compared to 48,120, and 144 h durations. In vitro viable mature oocytes showed higher IVF rates when fertilized with VS compared to ES and TS. The results showed that canine oocytes are good model for azoospermic patients

Effects of transrectal palpation with the fetal membrane slip technique for early pregnancy diagnosis on the proportion and type of associated pregnancy loss in dairy cattle

OBJECTIVE: To assess the effect of transrectal palpation (TRP) performed with the fetal membrane slip (FMS) technique for early pregnancy diagnosis on the proportion and type of associated pregnancy losses (PLs) in dairy cattle. ANIMALS: 580 healthy pregnant cattle. PROCEDURES: Data for artificially inseminated females with 1 or 2 viable embryos detected by transrectal ultrasonography (TRUS) at approximately 30 days of gestation were retrospectively assessed. Cattle were assigned to 1 of 2 groups on the basis of whether they did or did not undergo TRP once between 34 and 41 days of gestation (palpation and control group, respectively). At approximately 45 and 60 days of gestation, all cattle were reevaluated by TRUS; PL was categorized as type I (FMS detectable by TRP and TRUS-confirmed evidence of embryo or fetus degeneration and a functional corpus luteum) or type II (FMS undetectable by TRP and no TRUS-confirmed evidence of an embryo or fetus or of a functional corpus luteum). RESULTS: Of the 580 healthy pregnant cattle, 271 underwent TRP and 309 did not. In the palpation and control groups, PL occurred in 40 (14.8%) and 47 (15.2%) cattle, respectively. Among the palpation group\u27s PLs, 17 (43%) were type I and 23 (58%) were type II. Among the control group\u27s PLs, 27 (57%) were type I and 20 (43%) were type II. The prevalance and type of PL did not differ between groups. CONCLUSIONS AND CLINICAL RELEVANCE: TRP with the FMS technique for early pregnancy diagnosis did not increase the prevalence of PL in dairy cattle or alert the proportion of type I versus type II PL

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

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