H. Gustafsson

Repeat breeding in dairy heifers: follicular dynamics and estrous cycle characteristics in relation to sexual hormone patterns

Repeat breeding occurs at an incidence of 10% in the Swedish dairy cow population. Evidence is available for a hormonal asynchrony around estrus in repeat-breeder heifers (RBH). This asynchrony seems to be the underlying cause for a series of dysfunctions such as prolonged standing estrus and delayed ovulation, leading to fertilization failure. For further determinations of repeat-breeder estrous cycle characteristics, seven strictly selected RBH and six virgin heifers (VH) were studied during 3-7 consecutive cycles, with particular attention paid to the estrous period. Follicular dynamics were studied by ultrasonography and related to estrous behavior and pattern of sexual hormones (progesterone, estradiol-17beta, and LH) in peripheral circulation. Mean group data were compared and a classification model was designed. The most prominent findings for RBH were prolonged duration of estrus, delayed LH peak, prolonged lifespan of the preovulatory follicle, and a late postovulatory rise in plasma progesterone. There was also a strong tendency for peri-ovulatory suprabasal progesterone levels in RBH. It is suspected that these deviations cause changes in the microenvironment of the preovulatory follicle, negatively affecting the final maturation of the oocyte. The heterogeneity of the RBH group underlines the multifactorial cause of the repeat-breeder syndrome. The VH formed a homogenous group with data varying within physiological limits. A classification model based on three characteristic variables managed to identify 81% of the VH and 79% of the RBH correctly. Results from this study propose that some heifers have general, consistent problems in synchronizing estrous events, displayed as varying symptoms in the course of consecutive estrous cycles. These subfertile animals could be classified as repeat-breeders.

A SERUM-FREE, CELL-FREE CULTURE SYSTEM FOR DEVELOPMENT OF BOVINE ONE-CELL EMBRYOS UP TO BLASTOCYST STAGE WITH IMPROVED VIABILITY

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.

Effect of induced suprabasal progesterone levels around estrus on plasma concentrations of progesterone, estradiol-17β and LH in heifers

A controlled study was carried out to investigate the effects of suprabasal plasma progesterone concentrations on blood plasma patterns of progesterone, LH and estradiol-17beta around estrus. Heifers were assigned to receive subcutaneous silicone implants containing 2.5 g (n=4), 5 g (n=4), 6 g (n=3), 7.5 g (n=3) or 10 g (n=4) of progesterone, or implants without hormone (controls, n=5). The implants were inserted on Day 8 of the cycle (Day 0=ovulation) and left in place for 17 d. The time of ovulation was determined by ultrasound scanning. Blood was collected daily from Days 0 to 14 and at 2 to 4-h intervals from Days 15 to 27. Control heifers had the lowest progesterone concentrations on Days 20.5 to 21 (0.5 +/- 0.1 nmol L(-1)); a similar pattern was observed in heifers treated with 2.5 and 5 g of progesterone. In the same period, mean progesterone concentrations in the heifers treated with 6, 7.5 and 10 g were larger (P < 0.05) than in the controls, remaining between 1 and 2.4 nmol L(-1) until implant removal. A preovulatory estradiol increase started on Days 16.4 to 18.4 in all the animals. In the controls and in heifers treated with 2.5 and 5 g of progesterone, estradiol peaked and was followed by the onset of an LH surge. In the remaining treatments, estradiol release was prolonged and increased (P < 0.05), while the LH peak was delayed (P < 0.05) until the end of the increase in estradiol concentration. The estrous cycle was consequently extended (P < 0.05). In all heifers, onset of the LH surge occurred when progesterone reached 0.4 to 1.2 nmol L(-1). The induction of suprabasal levels of progesterone after spontaneous luteolysis caused endocrine asynchronies similar to those observed in cases of repeat breeding. It is suggested that suprabasal concentrations of progesterone around estrus may be a cause of disturbances oestrus/ovulation.

Sequential endocrine changes and behaviour during oestrus and metoestrus in repeat breeder and virgin heifers

Abstract The aim of the experiment was to study the oestrous behaviour and the peripheral blood plasma profiles of luteinizing hormone (LH), progesterone and the prostaglandin metabolite, 15-keto-13,14-dihydro-PGF2α, during oestrus and metoestrus in repeat breeder (RBH) and virgin heifers (VH). Ten animals of each category were utilized. The RBH had a history of at least three inseminations without conception, and the VH were sexually mature animals not previously inseminated or mated. Oestrous symptoms were recorded and blood collected from the onset of prooestrus to 7 days after oestrus. The animals were inseminated during oestrus and their embryos were collected by a nonsurgical technique 7 days after insemination. The morphology of the embryos was evaluated. The duration of oestrus was longer (P The result of the study indicates a hormonal imbalance in the RBH. The hormonal asynchronism starts before or early in oestrus, which presumably leads to a sequence of improper hormonal changes responsible for the elevated embryonic loss in repeat breeder animals.

Influence of perioestrous suprabasal progesterone levels on cycle length, oestrous behaviour and ovulation in heifers

In order to induce suprabasal plasma concentrations of progesterone after luteolysis and to determine their effect on oestrous behaviour and ovulation, heifers subcutaneously received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3) or 10 (n = 4) g of progesterone, or an empty implant (controls, n = 5) between days 8 and 25 of the cycle (ovulation designated Day 0). Growth of dominant follicles and time of ovulation were determined by ultrasound, and signs of oestrus were recorded and scored. Blood was collected at 2–4 h intervals from Days 15 to 27 and assayed for progesterone concentration. In all heifers, plasma concentrations of progesterone sharply decreased during Days 16–18. Control heifers had their lowest progesterone levels on Days 20.5 and 21, standing oestrus on Day 19.5 ± 0.4 (mean ± SEM), and ovulated on Day 20.7 ± 0.4. A similar pattern was observed in heifers treated with 2.5 and 5 g progesterone. Heifers treated with 6, 7.5 and 10 g of progesterone showed an extended (P < 0.05) interovulatory interval. Onset of prooestrus and time of maximum expression of signs of oestrus were not significantly different from those in controls. However, there was an absence of standing oestrus in most of the cases, signs of oestrus lasted longer (P < 0.05) and were weaker in intensity when doses increased. In these groups, the lowest progesterone concentrations were attained shortly after implant removal. Some heifers treated with 6 and 7.5 g of progesterone had standing oestrus and post oestrous bleeding as seen in the controls but ovulation occurred from Days 24.5 to 27. When plasma progesterone concentrations were over 1 nmol 1−1, disturbed oestrus and delayed ovulation occurred. The extended period of prooestrus and oestrus and delayed ovulation were similar to that described in cases of repeat breeding. It is suggested that suprabasal plasma concentrations of progesterone, after luteolysis, may lead to asynchrony between onset of oestrus and ovulation and consequently be a cause of repeat breeding in cattle.

Effect of ACTH-challenge on progesterone and cortisol levels in ovariectomised repeat breeder heifers

In order to investigate the potential influence of stress as a component of the repeat breeding syndrome, the adrenocortical capacity for steroid production was evaluated in ovariectomised dairy heifers. In repeat breeder heifers (RBH), marginally elevated plasma progesterone levels during oestrus, so-called suprabasal progesterone levels, have earlier been measured and are believed to impair fertility. The aim was to distinguish if this progesterone could be of extra-gonadal or in this case, adrenal origin. Baseline levels of plasma cortisol and progesterone were determined as well as the corresponding response after induced acute stress in the form of an adrenocorticotropin (ACTH)-challenge. Comparisons were made between strictly selected RBH, n=5 and virgin heifers (VH), n=5 of the Swedish Red and White breed. The heifers were used as their own pre-challenge controls in a 2-day trial. On the control day, saline was injected i.v. and on the treatment day, a synthetic analogue of ACTH (60 microg Synachten(R)). Via a jugular vein catheter, blood samples were collected every 30 min for 6 h each day of the experiment. Analyses for plasma progesterone and cortisol were made. RBH had a significantly higher (P<0.01) pretreatment baseline cortisol level (10.1+/-2.3 nmol l(-1)) than VH (2.6+/-0.2 nmol l(-1)). Moreover, the cortisol response after stimuli was stronger in RBH than VH, especially concerning total hormone production (P<0. 001), but there was also a tendency towards higher peak values (P=0. 06) and longer duration of significantly increased hormone concentrations (P=0.08). Progesterone concentrations, however, did not differ between the groups. Both baseline levels (P=0.25) and posttreatment production (P=0.45) were of the same magnitude in RBH and VH. In conclusion, the study could not confirm that suprabasal progesterone concentrations during oestrus in RBH derive from the adrenal glands. Still, apparent differences were found in adrenocortical activity when ovariectomised heifers, VH and RBH, were subjected to an ACTH-challenge. It is suggested that a sustained adrenal stimulation associated with environmental or social stress could be one factor in the repeat breeding syndrome.

The effect on luteolysis by intensive oral administration of flunixin granules in heifers

The objective of the study was to investigate whether oral administration of flunixin meglumine (FM) in the form of granules could prolong the functional life of the corpus luteum in heifers. Previous studies have shown that intensive, i.e. four times daily parental administration of FM can postpone luteolysis. Twelve heifers received an oral dose of 2.2 mg flunixin per kg body weight. Three dosing regimes were used; twice (n = 2), thrice (n = 4) and four times daily (n = 6). The 9-day-treatment period started on Day 14/15 of the oestrous cycle. Blood samples were collected twice daily during the entire experimental period. Frequent samples were withdrawn from Day 14/15 for 10 and 15 days in the control and treatment oestrous cycles, respectively, at 0600, 0800, 1000, 1200, 1400, 1600, 1800 and at 2000 h. The plasma was analysed for the content of the main metabolite of PGF2 alpha, i.e. 15-ketodihydro-PGF2 alpha, and progesterone. A control cycle preceded the treatment cycle so that each heifer acted as its own control. The length of the oestrous cycle was significantly increased when FM was administered thrice, from 18-22 days to 20-24 days (P < 0.05), and four times daily, from 18-21 days to 25-27 days (P < 0.01), but not in the twice daily dosing regime (one-way ANOVA). When FM was administered twice and thrice, luteolysis occurred during treatment. However, when the four times daily regime was used, luteolysis was obtained when the treatment had terminated. No changes in progesterone levels were recorded, although the luteal phase increased when the oestrous cycle length was prolonged. The number of PG-pulses decreased significantly, from 6-12 pulses to 0-3 pulses (P < 0.01), when FM was administered four times daily. A reduction was also observed when heifers received the drug thrice, but the decrease was not significant. The oral route of administration was found to be as effective as the parental one to affect the mechanism responsible for luteolysis in heifers. However, to inhibit and postpone luteolysis, administration of FM four times daily is a necessity. Our results show that oral administration of FM in the bovine species can be of value both in research as well as in the clinic, e.g. to support the luteal function.

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.

Supplemental feeding with glycerol or propylene glycol of dairy cows in early lactation—Effects on metabolic status, body condition, and milk yield

The objective of this field study was to evaluate the effect of supplemental feeding with glycerol or propylene glycol to dairy cows in early lactation on metabolic status, body condition and milk yield. In total, 673 newly calved cows from 12 commercial Swedish dairy herds were randomized to daily supplementation with 450 g of glycerol (GLY), 300 g of propylene glycol (PG), or nothing (control, CON). Supplements were fed twice daily from 0 to 21 d in milk (DIM) as a top dress on concentrates. For each cow, data on parity, breed, calving date, monthly test-day milk yield, and cases of diseases were collected. Blood samples were taken at approximately 2, 5, and 8 wk postpartum (pp) and analyzed for glucose, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), and insulin. Samples taken within 3 wk pp were also analyzed for insulin-like growth factor 1 (IGF-1). Measurements of body condition score (BCS) and heart girth (HG) were obtained at approximately 2 and 5 wk pp and at time of first insemination. The effects of supplemental feeding with GLY or PG on the plasma concentrations of glucose, NEFA, BHBA, insulin, and IGF-1, and BCS, HG, and occurrence of disease were analyzed. No differences in BCS or HG or in plasma concentrations of glucose, BHBA, NEFA, or IGF-1 were found between the control group and any of the treatment groups. Cows in the GLY group had lower plasma insulin concentrations during DIM 0 to 63 compared with group CON, but no difference in insulin was found between the PG group and the CON group. Cows supplemented with GLY had a higher milk yield (kg of milk and kg of energy-corrected milk) during the first 90 DIM. Cows in the PG group tended to yield more milk during the same period. No differences in the occurrence of diseases were seen between the groups. In conclusion, supplementation with GLY in early lactation did increase milk yield without a subsequent decrease of metabolic status, and supplementation with PG tended to do the same.

Characteristics of embryos from repeat breeder and virgin heifers.

The characteristics of 7-day-old embryos non-surgically collected from 35 repeat breeder heifers (RBH) and 24 virgin heifers (VH) were compared by repeated observations within each animal. A higher incidence of the embryos collected from the VH was classified as normal and had reached a more advanced developmental stage than embryos from the RBH. Nearly all VH yielded normal (N) embryos, but morphologically deviated (MD) or degenerated embryos (D) appeared occasionally in many VH. The RBH group contained three subgroups of animals. One group of RBH yielded a high percent of N embryos. A second group yielded mostly MD or D embryos with an occasional N embryo, and the third group only D embryos or no embryos. Heifers from which no embryos were recovered on day 7 yielded uncleaved ova, apparently retarded embryos or no embryos when slaughtered three days after insemination. It is concluded that retarded embryonic development may be a common factor for most RBH. The embryo morphology and the degree of retardation differ among animals and between oestrous periods in the same animal.