Renée Båge

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.

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.

Two different schemes of twice-weekly ovum pick-up in dairy heifers: effect on oocyte recovery and ovarian function

The aim of the present study was to compare two different schemes of twice-weekly ovum pick-up (OPU), continuous (C) and discontinuous (DC), with special emphasis on differences in oocyte yield and quality, estrous cyclicity, ovarian dynamics, and progesterone production. Subsequent to characterization of their normal estrous cycles (pre-OPU period), eight dairy heifers were subjected to 4 months of twice-weekly OPU under two different schemes: the DC (OPU restricted to Days 0-12 of the cycle) and the C schemes. Effects of the two different schemes on oocyte yield, quality, and in vitro competence, together with effects on ovarian dynamics and progesterone production, were monitored. The mean numbers of punctured follicles and recovered oocytes per session were slightly higher (not significant (n.s.)) using the DC scheme, but in total, similar numbers of oocytes were obtained. The quality of the oocytes as well as cleavage rate after in vitro fertilization of the oocytes did not differ between the two OPU schemes. There was no influence of a corpus luteum (CL) producing progesterone on the oocyte yield and quality, whereas the presence of dominant follicles appeared to decrease the number of recovered ooctyes. During the pre-OPU period, all heifers showed normal cyclicity. In the DC scheme, the heifers showed regular and normal cyclic activity throughout the puncture period, with one to two complete follicular waves during the interval from the last OPU to the next estrus. In the C scheme, the heifers occasionally revealed cyclicities with irregular interestrous intervals and weaker signs of estrus. No complete follicular waves were seen during the OPU period in this scheme. The CL developed from the ovulation of the preovulatory follicles in the DC scheme showed similar characteristics to the CLs of the pre-OPU period; however, the CL-like structures from the puncture of follicles, in both the DC and the C schemes, revealed a shorter life span and inferior competence in producing progesterone (P<0.05). The present results indicate that the DC OPU scheme, which allows animals to go into natural ovulation prior to the first OPU, does not affect their ovarian function, whereas the C OPU scheme does. Our study further demonstrates that an equal number of oocytes can be obtained with both schemes, but that fewer OPUs are needed when the DC scheme is applied.

Do Cytoplasmic Lipid Droplets Accumulate in Immature Oocytes from Over-Conditioned Repeat Breeder Dairy Heifers?

One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed.

Oocyte competence in repeat-breeder heifers: effects of an optimized ovum pick-up schedule on expression of oestrus, follicular development and fertility.

Repeat-breeder heifers (RBH) have been shown to present reproductive perturbations during spontaneous cyclicity, which affects oestrus and ovulation. Some of these disturbances (e.g. deviating hormone patterns) are also present during and after cycles of twice-weekly ovum pick-up (o.p.u.), performed according to an optimized schedule allowing normal oestrous cyclicity. In the present study, the effects of o.p.u. on oocyte competence in in vitro maturation (i.v.m.) and in vitro fertilization (i.v.f.) have been evaluated, as were the effects on expression of oestrus and fertility in five RBH (> or =4 artificial inseminations) and five virgin heifers (VH controls). In total, 269 RBH and 174 VH oocytes were scored for quality prior to i.v.m. and i.v.f. The number of follicles available for puncture was higher in RBH, but the oocyte recovery rate after o.p.u. was lower in RBH compared with VH controls and the recovered RBH oocytes were of lower quality, as judged by their appearance at retrieval. Confocal laser scanning and transmission electron microscopy of immature oocytes did not reveal any differences between RBH and VH control oocytes with respect to nuclear and mitochondrial status. However, after i.v.m., the cytoplasmic spatial reorganization of mitochondria and cortical granules was less advanced in RBH, which could contribute to the subfertility that defines the syndrome. Cleavage rates after i.v.f. were similar in RBH and VH controls. Subsequent to the o.p.u. period, in vivo fertility after controlled artificial insemination was comparable with field fertility rates in both RBH and VH.

OVARIAN FOLLICLE APOPTOSIS AT THE ONSET OF STANDING ESTRUS IN VIRGIN AND REPEAT-BREEDER DAIRY HEIFERS

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.

Pregnancy Rates in Repeat-breeder Heifers Following Multiple Artificial Inseminations during Spontaneous Oestrus

AbstractHormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P4 concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.SammanfattningDräktighetsresultat hos symptomlösa omlöparkvigor efter att de inseminerats upprepade gånger under spontan brunst. Hormonstörningar under brunsten, t ex suprabasala progesteronnivåer, och påverkat brunstförlopp med fördröjd ovulation har setts i samband med symptomlös omlöpning hos SRB-kvigor. Det är känt att hormonstörningar i sin tur kan störa äggledarfunktionen och påverka tillgången på befruktningsdugliga spermier vid ovulationstidpunkten. I det aktuella försöket inseminerades omlöparkvigor och normala kvigor med 6 h intervall från högbrunstens start till ovulation för att se om dräktighetsresultatet kunde påverkas positivt. Hormonanalyser visade att omlöparkvigorna hade suprabasala progesteronnivåer under brunsten och fördröjd preovulatorisk LH-frisättning, medan kortisolnivåerna var normala. Högbrunsten varade längre hos omlöparkvigorna, vilka därför inseminerades fler gånger än de normala kvigorna. Dräktighetsprocenten hos omlöparkvigorna steg till normala nivåer (60%), medan de normala kvigorna hade lägre dräktighetsprocent än väntat, vilket kan bero på negativ effekt av de frekventa inseminationerna. Resultaten från försöket visar att dräktighetsresultaten kan förbättras hos symptomlösa omlöparkvigor om de insemineras upprepade gånger tills spontan ovulation sker.

Deviant peri-oestrual hormone patterns affect the epithelium of the uterine tube in repeat-breeder heifers

In the bovine reproductive tract, the uterine tube is the critical site for a series of events required for fertilization and early embryonic development. In previous studies, a defined category of subfertile heifers, repeat-breeder heifers (RBH), has presented peri-oestrual disturbances (deviating hormone patterns and follicular dynamics) and uterine maternal-embryonic asynchrony. The present study aimed to investigate if tubal function was also affected, by determination of differences in the morphology of the tubal lining epithelium of RBH (n = 4) in comparison to controls (n = 6) during standing oestrus, studied by light and electron microscopy (SEM/TEM), and relate this to steroid hormone concentrations and receptor distribution in the target tissues. Tissue distribution of oestrogen receptor alpha (ERalpha) and progesterone receptor B (PRB) was quantified using immunohistochemistry. In particular, secretory cells differed in appearance between RBH and controls. The cells were less lumen protruding, microvilli were fewer and smaller and secretory granules in the apical cytoplasm were more numerous in RBH. Furthermore, the tubal epithelium was conspicuously coated with amorphous material. Morphological differences between categories were not explained hormonally or by steroid receptor distribution, except in two heifers from which uterine tubes were obtained after the luteinizing hormone (LH) surge. The isthmic PRB : ERalpha ratio was twice as high in the RBH than in the control. The deviating ultrastructure found in RBH, before and after the LH surge, might influence the tubal microenvironment with effects on gamete transport and final maturation and early embryonic development. The present study confirms that previously recorded perturbations in reproductive physiology in RBH are also manifested in the uterine tube, mainly by a deviating ultrastructure of the lining epithelium.

Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential.

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL-1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

Characterization of coagulase negative staphylococci from cases of subclinical mastitis in dairy cattle in Kampala, Uganda

BackgroundCoagulase negative staphylococci (CNS) are the most common pathogens leading to subclinical mastitis (SCM) in dairy cattle in Uganda. Coagulase negative staphylococci can vary between bacterial species in how they cause disease. The aim of the study was to characterize CNS, from cows with SCM in Uganda, at the species level.FindingsQuarter milk samples (n = 166) were collected from 78 animals with SCM. Bacteriological analyses were carried out at Makerere University, Kampala, Uganda and at the National Veterinary Institute (SVA), Uppsala, Sweden. The most common pathogens found in milk samples from cows with SCM were CNS (31.7%). Two species of CNS were found, S. epidermidis (85%) and S. haemolyticus (15%). Of the CNS isolates, 16/20 (80%) were positive for β-lactamase production (β+).ConclusionsIn milk samples from cows with SCM caused by CNS, S. epidermidis was most prevalent, followed by S. haemolyticus.