High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows.

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.

Effects of dexamethasone administration to diestrus cows on systemic progesterone, estrogen and uterine cyclooxygenase production.

Parenteral administration of dexamethasone to diestrus cattle can extend the length of the natural estrous cycle. In mice, dexamethasone has been shown to inhibit production of the second isozyme of the cyclooxygenase (COX) enzyme (a rate limiting enzyme in prostaglandin formation). Therefore, the purpose of this study was to determine the effect of dexamethasone on estrous cycle length and COX-1 and -2 production by the uterine endometrium of cyclic cattle. Nine crossbred beef cows that exhibited two previous normal estrous cycles were randomly assigned to two treatments; a control group administered intramuscular injections of vehicle, and a dexamethasone group administered 8 mg of dexamethasone (Azium, Schering Corp., Kenilworth, NJ). Both groups received twice daily injections on day 13-22 of the treatment cycle. Uterine endometrial biopsies were collected on days 16, 19 and 22 of the treatment cycle. Blood samples were collected daily on day 13-22 of the treatment cycle for plasma progesterone and estradiol concentrations.

Chromosomal analysis of mammalian oocytes matured in vitro with various culture systems.

The objective of this study was to evaluate oocyte maturation in vitro. Ten virgin CD-1 mice were used with 3 replications for in vitro with 4 different culture media. Media were minimal essential medium (MEM) with Earl's salt, Waymouth MB 752/1 (MB 752/1), BGjb medium (BGjb), and tissue culture medium-199 (TCM-199). The oocyte chromosomes were C-banded to enable an objective analysis of the chromosome abnormality and number. There was a percentage of blockage at metaphase I (M I), in matured oocytes in all culture media. Metaphase II (M II) was reached by 70.9 to 87.3% of oocytes in 4 different culture media. The frequencies of hyperploid M II oocytes were 0.0, 1.1, 2.8 and 2.6% for TCM-199, MEM, MB 752/1 and BGjb, respectively. A small proportion of oocytes was also found to be polyploid in 4 different culture media. There was an occurrence of premature centromere separation among oocytes. It was concluded that the chromosomes of the oocytes matured in vitro were not all in the normal condition (being at M II). The media used in this study for oocyte maturation caused maturation delay (being blocked at M I), premature centromere separation, polyploidy, and aneuploidy (such as, hyperploid, hypoploid).

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle.

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.

Effect of media substitutes on bovine granulosa cell function and proliferation during in vitro culture.

The development of a serum-free culture system for bovine granulosa cells that would allow for cellular proliferation without induction of steroidogenesis would provide researchers with an important in vitro tool for determining differentiation mechanisms during folliculogenesis. The objective of the present study was to determine the effect of a commercially prepared serum substitute and a medium supplement on proliferation and progesterone production by bovine granulosa cells. Granulosa cells were obtained by aspirating the follicular fluid of follicles 2 to 8 mm in diameter. For each experiment, growth curves to determine the proliferative and steroidogenic response of granulosa cells to several different medium additions were constructed. Cells were counted on d 1, 2, 4, 6, and 8 of culture to determine cell concentration and the media harvested to determine progesterone content. In Exp. 1, granulosa cells were seeded at an initial rate of 5.0 x 10(5) for 48 h in serum-supplemented medium then allotted to one of five treatments including medium alone or medium containing fetal bovine serum (FBS; 1%), Gibco BRL media supplement-x (GMS-X; 1%), fatty acid-free bovine serum albumin (FAF-BSA; 4 mg/mL), or SerXtend (5%). For Exp. 2 and 3, granulosa cells were plated in serum-supplemented medium for either 48 or 24 h and seeded at either 5.0 x 10(5) or 2.5 x 10(5) cells/flask, respectively, and cultured in different concentrations of SerXtend. All treatment media supported granulosa cell proliferation to some extent; the SerXtend treatment provided the highest proliferation rate at all concentrations above .3125%. Also, during the proliferative stage of the growth curve, cells in the SerXtend treatment produced lower amounts of progesterone compared with cells in the other treatments. In summary, granulosa cells may be propagated in vitro in a serum-free environment without inducing progesterone production.

Frozen-thawed cumulus-granulosa cells support bovine embryo development during coculture.

OBJECTIVE: To evaluate the ability of bovine cumulus-granulosa cells to survive cryopreservation and subsequently support bovine embryo development during coculture. DESIGN: In vitro-matured and -fertilized bovine embryos (two- to four-cell) were allotted randomly to one of three treatment groups: [1] control medium alone consisting of Medium 199 containing 10% fetal bovine serum and antibiotics, [2] cocultured on fresh bovine cumulus-granulosa cells in control medium, or [3] cocultured on frozen-thawed cumulus-granulosa cells in control medium. Embryo development was assessed on days 7 and 8 after IVF. RESULTS: Coculture improved embryo development on days 7 and 8 compared with the control group. However, embryo development on days 7 and 8 did not differ among coculture groups. CONCLUSIONS: Frozen-thawed cumulus-granulosa cells enhance embryo development similar to fresh cells during in vitro coculture.

The effects of different types of syringes on equine spermatozoa.

Control extender was incubated at 4 degrees C for 24 hours. Rubber or plastic syringe plungers were separately incubated in semen extender for 24 hours at 4 degrees C. Following incubation, the extender was stored at -20 degrees C until the time of semen collection. The treatments consisted of the following: Group A = equine semen plus control extender; Group B=equine semen plus extender incubated with rubber plungers and Group C=equine semen plus extender incubated in plastic plungers; Group D=equine semen plus control extended in rubber plunger syringes and Group E=equine semen plus control extender in plastic plunger syringer. Each group contained a 5-ml volume of semen and extender at a concentration of 1.0 x 10(8) sperm/ml. The number of live spermatozoa, percentage of progressively motile spermatozoa and rate of progressive motility were taken following collection and every 15 minutes for 1 hour following application of treatments. In experiment 2, treatments were allowed to incubate with semen for 45 minutes, then the extender was removed and was replaced with fresh extender. The rate of progressive motility and the percentage of progressively motile spermatozoa were taken immediately, at 45 minutes, and then every 15 minutes for 1 hour. In experiment 1, the number of live spermatozoa was not affected among the 5 groups. However, there was a decrease (P<0.01) in the rate of progressive motility and in the percentage of progressively motile spermatozoa in Group B compared with the remaining 4 treatment groups at 30, 45 and 60 minutes, with no differences noted when semen was held in syringes with a rubber or a plastic plunger. In experiment 2, the percentage of progressively motile spermatozoa increased after the addition of the control extender.

Intrauterine infusion of prostaglandin E2 and subsequent luteal function in cattle.

The objective was to evaluate the effect of intrauterine infusion of prostaglandin E2 (PGE2) on luteal function in cattle. Heifers and cows were randomly assigned after two normal estrous cycles to either PGE2 or control treatment groups. Females in Treatment A were infused with 1 mg of PGE2 once daily into the uterine horn ipsilateral to the corpus luteum between days 7-10 of the estrous cycle with a 0.25 ml plastic semen straw and an artificial insemination pipette. Females in Treatment B were similarly infused with 1 mg of PGE2 once daily in 20 ml of a carrier vehicle via a catheter on days 10 and 11 of the estrous cycle. Control animals were infused with the carrier vehicle using either a semen straw (Treatment C) or via a catheter (Treatment D) on the same days of the estrous cycle. Blood samples were collected daily to monitor plasma progesterone concentrations during the treatment period. Females infused with PGE2 on days 7-10 of the estrous cycle returned to estrus in a mean of 23.5 days (range 22-25 days) and were similar (P > 0.05) to those infused on days 10 and 11 which returned to estrus in 23.5 days (range 22-25 days). Animals similarly infused with carrier vehicle on the same days of the estrous cycle returned to standing estrus in 20.2 days (range 17-23 days). Plasma progesterone concentrations indicated an extended period of elevated progesterone concentrations in PGE2-treated animals compared with control animals. These results indicate that short term administration of PGE2 early in the estrous cycle may result in extended luteal maintenance.

Coculture of in vitro fertilized bovine embryos with oviductal epithelial cells originating from different stages of the estrous cycle.

Bovine embryos derived from in vitro fertilization procedures were cocultured in vitro with oviductal cells obtained from heifers between d 4 and 6 or d 14 and 16 of the estrous cycle. In addition, proteins secreted by oviductal cells isolated between d 4 and 6 or d 14 and 16 of the cycle were monitored. Embryos (2- to 4-cell) were incubated in Tissue Culture Medium-199 with 10% fetal bovine serum with or without oviductal cells at 39 degrees C for 10 d following in vitro insemination. There were more morulae, blastocysts, and hatched blastocysts following coculture with oviductal cells than with culture in medium alone. However, no differences were noted in embryo development following coculture with oviductal cells obtained between d 4 and 6 or d 14 and 16 of the estrous cycle. Also, no differences were detected in the amount of [35S]methionine-labeled proteins secreted by oviductal cells isolated from different days of the estrous cycle. These results indicate that oviductal epithelial cells isolated from early and late luteal phases of the estrous cycle will effectively support early embryonic development following prolonged in vitro culture.

The use of image analysis to evaluate the development of uterine and oviduct epithelial cells during in vitro culture. A potential quality assurance procedure for in vitro laboratories.

The objective of this study was to establish selection criteria for morphologic assessment of cell quality using a computer image-analysis system. Uterine and oviduct epithelial cells were isolated from the reproductive tracts of cyclic cows using a trypsin solution. Harvested cells were cultured in Tissue Culture Medium-199 with 10% fetal bovine serum and maintained at 37 degrees C with 5% carbon dioxide in air. Subcultures of different confluent monolayers were established using a weak trypsin solution. Morphologic assessment of cell integrity and viability were made visually, with electron microscopy and image analysis. After morphologic assessment, cells were assigned quality scores. Evaluations were conducted on cells during primary culture and following the first and third subpassages. Image-analysis evaluation was conducted on cells using a silicon-intensified target camera and computer-based software. Gray-level density was determined on cells from each flask. There was no difference (P greater than .05) between sample replicates, indicating repeatable measurements were obtained with the image-analysis system. In addition, there was no significant difference in parameters measured between uterine and oviduct cells; therefore, data from both cell types were combined for further statistical comparisons. Gray-level density values for combined uterine and oviduct cells during primary culture and following the first and third subpassages were as follows: 139.7, 154.5, and 173.3, respectively. There was an increase (P less than .05) in gray-level density values with each subpassage of the cell populations. Furthermore, gray level was influenced (P less than .01) by cell quality for combined uterine and oviduct cell populations. These results indicate that epithelial cell quality during in vitro culture may be effectively determined using image analysis. This approach should not be overlooked in establishing quality control measures for uterine and oviduct cells before mammalian embryos are cocultured in vitro.

Evaluating an in vitro culture system of bovine uterine and oviduct epithelial cells for subsequent embryo co-culture.

Three experiments were conducted to evaluate the effects of culture medium and incubation temperature on bovine uterine and oviduct epithelial cell growth, so that the most efficient combination could then be used to develop a co-culture system for bovine embryos. In the first experiment, uterine and oviduct epithelial cells at either the second or third subpassage were incubated for 8 days at 37 degrees C with 5% CO2 in Tissue Culture Medium-199, CMRL-1066, Minimal Essential Medium, Ménézo's B2 or Ham's F-12 medium. In addition to plotting growth curves of cell populations, the cell cycle was monitored for 8 days by flow cytometry. Uterine and oviduct epithelial cells incubated in CMRL-1066 exhibited the highest growth rates during the 8-day culture period. However, there were no differences in cell cycle analysis among treatment groups during the incubation period. In the second experiment, CMRL-1066 medium was used to evaluate growth and proliferation of uterine and oviduct epithelial cells incubated at 37 degrees C or 39 degrees C; temperature had no significant effect on growth rates or proliferation rates for either uterine or oviduct cells during the 8-day incubation. In the third experiment, the more promising culture media for epithelial cell culture studies were chosen for in vitro maturation and subsequent in vitro fertilization (IVF) of bovine oocytes. Early cleavage-stage embryos produced by IVF procedures were subsequently cultured in vitro for 7 days in medium alone or with oviduct epithelial cells. In this study, the culture medium did not influence fertilization or cleavage rates. However, more embryos co-cultured with oviduct epithelial cells were considered viable after 7 days of incubation compared with embryos incubated in medium alone. These results indicate that various incubation conditions can influence the growth of bovine uterine and oviduct epithelial cells in vitro. However, in spite of changes in cell growth patterns, there does not appear to be a change in their embryotropic capabilities in vitro.

Effect of feeding various levels of sodium zeolite A on milk yield, milk composition and blood profiles in thermally stressed Holstein cows.

Mid-lactation Holstein cows (n = 48) were equally and randomly assigned to one of four feeding treatments of sodium zeolite-A (SZA). SZA was mixed in a grain mixture (50:50 grain to forage ratio) of 0% (control), 0.5%, 1.0% and 1.5% SZA on a dry matter intake basis. Cows were fed alfalfa hay in the first phase and corn silage in the second phase of the study as roughage sources. Milk samples were taken three times weekly (am and pm) and analyzed for milk fat, protein and lactose with blood profiles conducted from samples collected weekly. SZA significantly (P less than .05) increased feed intake at all three levels for both diets. Milk yield was significantly (P less than .05) greater in the alfalfa diet. However, milk fat percent and percent protein were greater (P less than .05) in the corn silage diet. The addition of SZA to the corn silage diet increased (P less than .05) milk fat percent at the 1.0% level and milk protein at the 1.5% level. Calcium in milk was significantly (P less than .01) increased and respiration rates significantly lowered (P less than .05) in both diets at the 1.0% level. Serum calcium was higher (P less than .05) at the 1.0 and 1.5% level in the hay diet and the 1.5% level in the corn silage diet. Also, serum glucose and alkaline phosphate levels were significantly (P less than .05) higher in the corn silage diet.

Effects of stage of the bovine oestrous cycle on in-vitro characteristics of uterine and oviductal epithelial cells.

The objective of this experiment was to evaluate the effects of stage of the bovine oestrous cycle on in-vitro morphology, growth and monolayer foundation of uterine and oviductal epithelial cells. Epithelial cells were isolated from the uterus and oviducts collected from cyclic cattle on the day of oestrus (Treatment A), and between days 4 to 6 (Treatment B), days 8 to 10 (Treatment C) and days 14 to 16 (Treatment D) of the oestrous cycle. The morphological development, per cent cell viability and cell attachment were evaluated during primary culture and after the first and third subpassages. The highest per cent cell viability and cell attachment during primary culture, respectively, were noted in Treatment B for both uterine (87.7 and 87.5%) and oviductal (88.4 and 87.2%) cell populations. Uterine epithelial cell populations in Treatments C and D, respectively, had the lowest viability (76.5 and 68.8%) and attachment (10.8 and 10.5%) during primary culture. There were marked improvements in cell viability and cell attachment following the first subpassage (P less than 0.001) compared with primary cultures for both uterine and oviductal cells. These results indicate that the stage of the oestrous cycle has dramatic effects on uterine and oviductal epithelial cell morphology and developmental patterns during primary in-vitro cultures. The stage of the oestrous cycle when cells are collected may be more important than was once realized when culturing early stage embryos in vitro.

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle.

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.

The refractometer index as a correction factor for urinary estradiol in rhesus females.

Refractometer indexes were standardized and found to be highly correlated (r = 0.831) to actual creatinine levels. Urinary estradiol corrected by creatinine levels and refractometer indexes were found to be highly correlated (r = 0.857). Predicted ovulation was the same day in 86% of the ovulatory cycles predicted by creatinine and refractometer corrected estradiol levels. Refractometer indexes may be used in place of creatinine levels to correct for urine concentration fluctuations when predicting ovulation in the rhesus female.

An efficient procedure for manual platelet counting.

Blood samples were collected by jugular venipuncture from 15 dairy heifers and the blood platelets were counted by manual methods. The platelets were found to be uniformly distributed across the rows, columns and sides of a Neubauer haemocytometer, and it was shown that counting any 10 squares on either side of the haemocytometer and multiplying by a constant factor would accurately predict the total platelet count. This procedure would greatly reduce the time required to count large numbers of platelets per sample, and reduce errors due to the fatigue associated with counting large numbers of samples.

Gonadotropin releasing hormone therapy in functional infertility of dairy cattle.

Effects of gonadotropin-releasing hormone (GnRH) on conception rate was tested in 379 repeat-breeders in nine large dairy herds in Louisiana. Cattle with three or more services were treated intramuscularly with GnRH at the time of artificial insemination. The conception rate for the repeat-breeders treated with GnRH was significantly greater than for the controls (56 vs 40%). Furthermore, repeat-breeders that were treated with GnRH for two consecutive times at insemination resulted in a 53% increase in conception rate over the controls.

Cortisol response in heifers to artificial insemination, natural mating, and no mating at estrus.

Eight mixed-breed dairy heifers were used in a random block design to study the stress-producing effect of various types of mating on heifers. Treatments consisted of artificial insemination, natural mating, or not mating at estrus. Stress was evaluated by measuring blood plasma cortisol. Blood samples were collected by indwelling jugular catheters at 60 and 30 min before treatment; immediately before treatment (0 min); and 5, 15, 30, 60, 120 and 180 min after treatment. Means for cortisol were not significant for treatment or treatment across time. Mean cortisol response to the artificial insemination, natural mating and no mating treatments were 5.36, 8.05 and 5.56 ng/ml, respectively. These results suggest that the use of artificial insemination does not impose and added stress at estrus.

Changes of aldosterone in blood serum of dairy cattle during estrous cycle.

Blood samples were taken daily from five lactating cows and five open heifers beginning with the 1st day postestrus and continuing until the animal was observed in standing estrus. Means of aldosterone in blood serum were 102.6 +/- 5.1 pg/ml for lactating cows and 94.1 +/- 5.1 pg/ml for open heifers. Age and lactation did not appear to be major factors affecting circulating aldosterone in the bovine species. Effect of day of the estrous cycle on aldosterone was quadratic. Concentrations of aldosterone for lactating cows started at 92.3 pg/ml on day 1 of the estrous cycle, peaked at 125.5 pg/ml on day 8, then declined steadily to a low of 43.2 pg/ml on day 20. There was no significant interaction of group by day. During the estrous cycle aldosterone appeared to be related to development of corpus luteum and progesterone. Sodium in blood serum was higher during estrus for lactating cows than open heifers for linear, quadratic, and cubic effects. Although not correlated with aldosterone, sodium exhibited similar cyclic patterns throughout the estrous cycle. Responses between sodium and aldosterone appeared delayed during estrus.