Morphological quality of oocytes and blood plasma metabolites in repeat breeding and early lactation dairy cows.

The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea.

Myosin heavy chain pattern in the Akhal-Teke horses.

This study investigates the myosin heavy chain (MyHC) isoform composition in the gluteus medius muscle of the Akhal-Teke horses using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Fifteen horses aged between 1.5 and 23.5 years were used in this study and divided into three age groups: 1.5 to 4 (n = 6), 9 to 13 (n = 5) and 18.5 to 23.5 years (n = 4). The average content of the MyHC I isoform was 11.72 ± 1.07% (variation between individuals: 7.09% to 20.14%). The relative content of the MyHC IIa and IIx isoforms was subsequently 38.20 ± 1.46% (30.73% to 48.78%) and 50.07 ± 1.10% (43.8% to 56.78%) from the total MyHC. The MyHC pattern in the skeletal muscles of the Akhal-Teke horses shows that the muscles of these horses have a high capacity both for endurance and speed.

The differential transcriptome and ontology profiles of floating and cumulus granulosa cells in stimulated human antral follicles.

Communication between various ovarian cell types is a prerequisite for folliculogenesis and ovulation. In antral follicles granulosa cells divide into two distinct populations of mural and cumulus granulosa cells (CGC), enveloping the antrum and surrounding the oocyte, respectively. Both cell types, with the mural compartment in excess, contribute to the floating granulosa cell (FGC) population in the follicular fluid. The aim of this study was to compare the transcriptomes of FGC and CGC in stimulated antral follicles obtained from 19 women undergoing IVF-ICSI procedure. FGC were obtained from follicular fluid during the follicle puncture procedure and CGC were acquired after oocyte denudation for micromanipulation. Gene expression analysis was conducted using the genome-wide Affymetrix transcriptome array. The expression profile of the two granulosa cell populations varied significantly. Out of 28 869 analysed transcripts 4480 were differentially expressed (q-value < 10(-4)) and 489 showed > or =2-fold difference in the expression level with 222 genes up-regulated in FGC and 267 in CGC. The transcriptome of FGC showed higher expression of genes involved in immune response, hematological system function and organismal injury, although CGC had genes involved in protein degradation and nervous system function up-regulated. Cell-to-cell signalling and interaction pathways were noted in both cell populations. Furthermore, numerous novel transcripts that have not been previously described in follicular physiology were identified. In conclusion, our results provide a solid basis for future studies in follicular biology that will help to identify molecular markers for oocyte and embryo viability in IVF.

Pregnancy percentage following deposition of sex-sorted sperm at different sites within the uterus in estrus-synchronized heifers.

Our objective was to assess the effect on heifer pregnancy rate of deposition at three sites within the uterus of frozen-thawed sex-sorted sperm at a fixed time after estrus synchronization. Estrus was synchronized in 209 heifers by administration of PGF2a 14 days apart. At 80-82 h after the second PGF2a injection, X-chromosomes bearing fractions of semen with 2.2 x 10(6) sperm in insemination dose were used for single insemination into the uterine body (UB-AI, n=91) or for intracornual deposition in the middle of the uterine horn (MH-AI, n=57) or close to the utero-tubal junction (UTJ-AI, n=61). The overall pregnancy rate was 43.1%. Pregnancy rates did not differ (P>0.05) among sites of sperm sperm deposition, between the two farms at which the heifers were kept or between the two bulls producing the semen. Within UB-AI, MH-AI and UTJ-AI treatments, pregnancy rates were 41.8%, 49.1% and 39.3%, respectively (P>0.05). Pooled across classes for deposition site, pregnancy rate was 25.1% higher (P<0.01) for heifers showing strong signs of estrus than for heifers showing weak signs of estrus (45.9 versus 20.8%, respectively). Embryonic and fetal loss from diagnosis of pregnancy to term and at calving equalled 5.6%. Of 88 calves of identified sex, 93.2% were female. In conclusion, pregnancy rates of heifers did not differ significantly following deposition of 2.2 x 10(6) sex-sorted sperm 80-82 h after the second PGF2a injection near the utero-tubal junction, in the middle of the horn or into the uterine body.

Changes in semen quality in Estonian Holstein AI bulls at 3, 5 and 7 years of age.

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.

Low semen dose intracornual insemination of cows at fixed time after PGF2alpha treatment or at spontaneous estrus.

The numbers of spermatozoa per insemination and the site of semen deposition in the uterine horn appear to interact to influence pregnancy rate. In two experiments, the effect of a single low dose (2 x 10(6) spermatozoa) intracornual insemination (LD-ICI) on bovine pregnancy rate was compared with that of intracornual (SD-ICI) and conventional (SD-AI) inseminations of 40 x 10(6) spermatozoa. In Experiment 1, 157 cows were treated twice with PGF(2)alpha at a 14-day interval and inseminated at a fixed time (80-82 h) after the second PGF(2)alpha injection using LD-ICI (n=44), SD-ICI (n=61) or SD-AI (n=52). In LD-ICI and SD-ICI groups, semen was deposited in the horn ipsilateral to the ovulatory follicle close to the utero-tubal junction (LD-ICI-UTJ, n=33 and SD-ICI-UTJ, n=41) or in the middle part of the horn (LD-ICI-MH, n=11 and SD-ICI-MH, n=20). Pregnancy rates after LD-ICI-UTJ, LD-ICI-MH, SD-ICI-UTJ and SD-ICI-MH were 27%, 27%, 39% and 35%, respectively (P>0.05). The total pregnancy rate after LD-ICI (27%) did not differ (P>0.05) from that after SD-ICI (37%) or SD-AI (34%). In Experiment 2 (field trial), 362 cows were allotted, at spontaneous estrus, to LD-ICI-UTJ (n=86), LD-ICI-MH (n=97) or SD-AI (n=179). Pregnancy rates after LD-ICI and SD-AI were 47% and 45%, respectively (P>0.05). After LD-ICI-UTJ, the pregnancy rate (54%) did not differ significantly (P>0.05) to that obtained after LD-ICI-MH (41%) and after SD-AI (45%). The results of the study show that the single intracornual insemination of cows with 2 x 10(6) spermatozoa at fixed time, 80-82 h after the second PGF(2)alpha injection or at spontaneous estrus resulted in similar pregnancy percentage as intracornual and conventional inseminations with 40 x 10(6) spermatozoa per semen dose. With intracornual insemination using low or standard dose of spermatozoa, the pregnancy rates were not significantly affected by the exact site of semen deposition in the uterine horn, near the utero-tubal junction or in the middle part.

Fixed time deep intracornual insemination of heifers at synchronized estrus.

The aim of the study was to determine the efficiency of single fixed time deep intracornual insemination using 2 x 10(6) spermatozoa compared with single standard dose deep intracornual insemination and single and dual standard dose (40 x 10(6)) uterine body (conventional) insemination in heifers at synchronized estrus. Estrus was synchronized in 275 virgin heifers by administration of two doses of PGF(2)alpha 14 days apart. Deep intracornual inseminations with low (ICI-LD1, n=102) and standard (ICI-SD1, n=56) dose of semen and the single standard dose conventional inseminations (AI-SD1, n=66) were performed 80-82 h after the second PGF(2)alpha treatment. Ultrasonography was used to identify the first dominant (presumed ovulatory) follicle, and semen was deposited either close to the utero-tubal junction (n=69 in ICI-LD1 and n=23 in ICI-SD1) or in the middle part of the uterine horn (n=28 in ICI-LD1 and n=28 in ICI-SD1) ipsilateral to the ovary bearing the first dominant follicle. The dual standard dose conventional inseminations were performed 72 and 96 h after the second PGF(2)alpha treatment (AI-SD2, n=51). The pregnancy rate in the ICI-LD1 group (68.0%) did not differ significantly (P>0.05) from the ICI-SD1 group (56.9%) or the AI-SD2 group (65.9%) and was significantly higher (P<0.05) than in the AI-SD1 group (54.2%). The site of intacornual deposition of semen, near the utero-tubal junction or in the middle of the horn, had no effect on the pregnancy rate. The pregnancy rate in all the groups was not affected by the intensity of expression of estrous signs.

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media.

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 microg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 microg/ml heparin, 20 microM D-penicillamine, 10 microM hypotaurine and 1 microM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 microg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS - TCM 199 supplemented with 10 mg/ml BSA and ITS (5 microg/ml insulin, 5 microg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM - bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).

Viability of fresh and frozen-thawed biopsied bovine embryos.

Bovine embryos were biopsied using a simplified splitting technique and frozen-thawed according to a standard method with glycerol as cryoprotectant. The viability of fresh and frozen-thawed biopsied and intact embryos were evaluated after in vitro culture, by means of fluorescence test or following transfer to recipients. The survival rates after in vitro culture of fresh intact and biopsied embryos and of frozen-thawed intact and zona free embryos were not significantly different (70%, 60%, 68% and 52%, respectively), but significantly reduced for biopsied frozen-thawed embryos (16%) (p < or = 0.05). The pregnancy results after transfer of biopsied frozen-thawed embryos were also significantly lower (8%) compared to fresh biopsied embryos (39%) (p < or = 0.05). Both intact and biopsied embryos fluoresced after incubation with diacetylfluorescin but with higher intensity for the intact embryos. It is suggested that the reduced survivability for the frozen-thawed biopsied embryos might be caused by combined effects of the loss of the zona pellucida and the reduction of cells as a result of the simplified biopsy technique. It is concluded that improved biopsy and/or freezing techniques must be used if biopsied embryos have to be frozen.