A. Van Soom

Reduced Fertility in High-yielding Dairy Cows: Are the Oocyte and Embryo in Danger? Part I The Importance of Negative Energy Balance and Altered Corpus Luteum Function to the Reduction of Oocyte and Embryo Quality in High-yielding Dairy Cows*

Fertility in high yielding dairy cows is declining, and there is increasing evidence to presume that oocyte and embryo quality are major factors in the complex pathogenesis of reproductive failure. In this report we present an overview of possible mechanisms linking negative energy balance (NEB) and deficiencies in oocyte and embryo developmental competence; specifically, in the high producing dairy cow. Changes in follicular growth patterns during a period of NEB can indirectly affect oocyte quality. The endocrine and biochemical changes, which are associated with NEB, are reflected in the microenvironment of the growing and maturing female gamete, and likely result in the ovulation of a developmentally incompetent oocyte. Even after an oocyte is successfully ovulated and fertilized, a full-term pregnancy is still not guaranteed. Inadequate corpus luteum function, associated with reduced progesterone, and probably also low insulin-like growth factor concentrations, can cause a suboptimal microenvironment in the uterus that is incapable of sustaining early embryonic life. This may partly account for the low conception rates and the high incidence of early embryonic mortality in high yielding dairy cows.

Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part II. Mechanisms linking nutrition and reduced oocyte and embryo quality in high-yielding dairy cows.

Dairy cow fertility has been declining during since the mid-80s and this has given rise to numerous scientific studies in which important parts of the pathogenesis are elucidated. Reduced oocyte and embryo quality are acknowledged as major factors in the widely described low conception rates and in the high prevalence of early embryonic mortality. Apart from the importance of the negative energy balance (NEB) and the associated endocrine and metabolic consequences, there is a growing attention towards the effect of the milk yield promoting diets which are rich in energy and protein. Starch-rich diets can improve the energy status and thus the ovarian activity in the early postpartum period but the oocyte and embryo quality can suffer from such insulinogenic diets. Supplementation of dietary fat has a similar dual effect with a beneficial stimulation of the ovarian steroid production while the oocyte and the embryo display an altered energy metabolism and excessive lipid accumulation. High-protein diets can elevate the ammonia and urea concentrations in the blood, leading to changed intrafollicular, oviductal and uterine environments. Oocytes and embryos are highly sensitive to such changes in their microenvironment, possibly leading to a disturbed maturation, fertilization or early cleavage. Several nutrition-linked mechanisms, through which oocyte and/or embryo quality can be affected in modern dairy cows, well after the period of NEB, are proposed and comprehensively reviewed in the present report.

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows n=30 and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6-8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, beta-hydroxybutyrate (beta-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, beta-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), beta-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.

Compaction rate of in vitro fertilized bovine embryos related to the interval from insemination to first cleavage

A study was conducted on early cleavage divisions and timing of compaction in bovine preimplantation-stage embryos. Zygotes were produced using conventional in vitro maturation and fertilization procedures. Twenty hours post insemination, the zygotes were denuded and cultured with oviduct epithelial cells in B2 medium + 10% estrous cow serum. Starting at 24 hours post insemination, the embryos (n=657) were evaluated every 6 hours and then were put into different co-culture drops according to their cell number. Starting from 78 hours post insemination, the cleavage rate was evaluated every 12 hours. Embryos were stained with Hoechst 33342 at the compacted morula stage or when they were degenerated, at 162 hours post insemination. Developmentally capable embryos were characterized by a rapid cleavage rate in the first 3 cell cycles and by an extended 8- to 16-cell stage. Peak concentrations of 2-, 4-, 8- and 16-cell stages emerged at 36, 42, 60 and 102 hours post insemination, respectively. Compaction did not occur until 126 hours post insemination. The rate of compaction was significantly higher in embryos that were at the 2-cell stage before or at 36 hours post insemination (P < 0.05). The mean cell numbers of compacted morulae that were identified at 126 and 138 hours post insemination were 30.9 +/- 6.8 and 31.6 +/- 7.7, respectively. These results indicate that developmentally capable bovine embryos reach the 2-cell stage at 36 hours post insemination, and that they become compacted at the 32-cell stage, which usually occurs between 126 and 138 hours post insemination.

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

Diseases in swine transmitted by artificial insemination : An overview

Artificial insemination (AI) of swine is widely practiced in countries with an intensive pig production. It is a very useful tool to introduce superior genes into sow herds, with minimal risk for disease transmission. However, the impact of semen that is contaminated with pathogens can be enormous. Most of the micro-organisms that have been detected in boar semen are considered non-pathogenic, but some are known pathogens (e.g. porcine reproductive and respiratory syndrome virus) that can cause major economic losses. Microbial contamination of semen can be due to systemic and/or urogenital tract infections of the boar, or can occur during collection, processing and storage. It can result in reduced semen quality, embryonic or fetal death, endometritis and systemic infection and/or disease in the recipient female. Conventional techniques for isolation of bacteria and viruses from the semen do not always provide optimal results for various reasons, including lack of sensitivity and speed of testing, and difficult interpretation of the outcome. More recently, PCR tests are commonly used; they have a high sensitivity, the outcome is quickly obtained, and they are suitable for monitoring a large number of samples. The best strategy to prevent AI-transmitted diseases is to use boars that are free of specific pathogens, to monitor the animals and semen regularly, and to maintain very high biosecurity. Additional measures should be directed at treating semen with appropriate antimicrobials, and at reducing contamination during semen collection, processing, and storage.

Embryonic mortality and embryo–pathogen interactions

Embryonic mortality (EM) has a substantial impact on the fertility of domestic animals. Most of the embryonic losses occur during the first days after fertilization and during the process of implantation. Causes of EM can be divided into infectious and non-infectious categories. Primary attention has often been given to infectious agents but non-infectious causes probably account for 70% or more of the cases of embryonic death. Infection of the embryonic environment can be caused by specific and non-specific uterine pathogens. Specific uterine infections are caused by a number of viruses, bacteria and protozoa that enter the uterus by the haematogenous route or via the vagina. Non-specific pathogens are mainly bacteria that enter the uterus by ascending infection. Uterine pathogens may cause EM by changing the uterine environment (endometritis) or by a direct cytolytic effect on the embryo. Non-infectious causes of EM such as chromosomal aberrations, external factors (e.g., high ambient temperature and nutritional factors) and maternal factors (e.g., hormonal imbalances and age) are multifactorial and difficult to diagnose.

The effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality

BACKGROUND Obesity is associated with female reproductive abnormalities. Hyperlipidaemia might alter the embryonic micro-environment and potentially result in reduced fertility. We aimed to induce hyperlipidaemia nutritionally and investigate the consequences of hyperlipidaemic culture conditions on bovine in vitro embryo development, embryo quality and gene expression patterns. METHODS Bovine zygotes (n = 1545) were cultured in synthetic oviductal fluid medium supplemented with serum from heifers (n = 3), each fed three successive dietary treatments: (i) control serum, following a hay-based diet, (ii) hyperlipidaemic serum, following a carbohydrate and protected palm-oil-rich diet (FatCh) or (iii) hyperlipidaemic serum, following a protected palm-oil-rich diet (Fat). Blastocysts were evaluated for development, cell count, picnotic and mitotic indexes and cryotolerance. Selected mRNA transcripts were measured by quantitative RT-PCR. RESULTS FatCh and Fat diets approximately doubled the total cholesterol concentrations, compared with controls (167.1 +/- 11.9, 150.0 +/- 12.8 versus 83.4 +/- 13.7 mg/dl, respectively, P < 0.05), and fatty acid concentrations (8146.60 +/- 214.61, 6935.56 +/- 1081.04 versus 3944.0 +/- 425.07 micromol/l, respectively, P < 0.05). Supplementation of culture media with FatCh and Fat serum significantly reduced blastocyst rates, compared with controls (27.8, 23.4% versus 36.2%, respectively, P < 0.01), total cell number (103.3 +/- 30.1, 95.6 +/- 28.2 versus 146.9 +/- 34.2, respectively, P < 0.01), mitotic index (1.3 +/- 1.1, 1.7 +/- 2.4 versus 3.6 +/- 2.2%, respectively, P < 0.01) and hatching rates after vitrification (20.4, 13.8 versus 35.7%, respectively, P = 0.03). Embryos in FatCh and Fat groups exhibited significantly higher mRNA levels for genes related to apoptosis and metabolism, compared with controls. CONCLUSIONS This combined in vivo and in vitro model indicates that the exposure of preimplantation embryos to hyperlipidaemic conditions may result in reduced embryo quality and developmental potential, possibly resulting in poorer fertility.

Biochemical changes in the follicular fluid of the dominant follicle of high producing dairy cows exposed to heat stress early post-partum

High yielding dairy cows experience a negative energy balance (NEB) early post-partum and it was hypothesized that this may be aggravated under summer heat stress (HS) conditions. In this study, which was performed in Egypt, 20 Holstein cows were followed during summer (n=10) and winter (n=10) seasons. All cows were multiparous and kept at the same herd. Blood was sampled from each cow starting 1 week before the expected calving date and then at 1-week intervals until week 6 post-partum. From week 2 to 6 post-partum follicular fluid was collected through transvaginal follicular fluid aspiration at 6 days intervals. Ambient air temperature (AT) and relative humidity (RH) were recorded and temperature-humidity index (THI) was calculated as well. Respiration rate (RR), rectal temperature (RT), and body condition score (BCS) were recorded for each cow at the time of blood sampling. Concentrations of glucose, insulin like growth factor-1 (IGF-1), non-esterified fatty acids (NEFA), urea and total cholesterol (TC) were measured in each blood and follicular fluid sample. All the cows showed a significantly higher RR and RT in summer (95.5+/-1.1 and 39.88+/-0.06, respectively) than in winter (43.89+/-0.61 and 38.94+/-0.07, respectively) (P<0.001). Body condition score loss during the early post-partum period was higher in summer than in winter (1.1+/-0.07 vs. 0.85+/-0.06 point, respectively) (P<0.001). The average dominant follicle diameter was significantly lower in summer than in winter during the period of negative energy balance (11.6+/-0.7mm vs. 15.3+/-1.2mm, respectively) (P<0.01). Under summer heat stress, the concentrations of glucose (2.98+/-0.07 and 2.19+/-0.04mmol/L), IGF-1 (106.7+/-2.9 and 99.0+/-3.4ng/ml) and TC (137.3+/-5.3 and 62.2+/-5.1mg/dl) in blood and FF, respectively, were significantly lower than winter concentrations by (0.17+/-0.03mmol/L, P<0.001 and 0.26+/-0.06mmol/L, P<0.001), (12.3+/-3.6ng/ml, P<0.001 and 9.0+/-2.7ng/ml, P<0.001) and (20.7+/-1.8mg/dl, P<0.001 and 7.3+/-1.1mg/dl, P<0.01), respectively. However, the concentrations of NEFA (0.68+/-0.14 and 0.22+/-0.02mmol/L) and urea (9.27+/-0.34 and 9.96+/-0.25mmol/L) in blood and FF, respectively, were significantly higher in summer compared to winter (0.50+/-0.08mmol/L, P<0.001 and 0.20+/-0.02mmol/L, P<0.001) and (8.77+/-0.23mmol/L, P<0.05 and 8.96+/-0.29mmol/L, P<0.001), respectively, throughout the experimental period. The results of the present study indicate that heat stress early post-partum aggravates NEB in high yielding dairy cows, reduces BCS, dominant follicle diameter and alters the biochemical concentrations in the follicular fluid of the dominant follicle which may result in inferior oocyte and granulosa cell quality and hence poorer fertility.