Dermot G. Morris

Embryonic and Early Foetal Losses in Cattle and Other Ruminants

Embryo survival is a major factor affecting production and economic efficiency in all systems of ruminant milk and meat production. For heifers, beef and moderate yielding dairy cows, does and camelids it appears that fertilization generally lies between 90% and 100%. In high-producing dairy cows there is a less substantive body of literature, but it would appear that it is somewhat lower and perhaps more variable. In cattle, the major component of embryo loss occurs before day 16 following breeding with some evidence of greater losses before day 8 in high-producing dairy cows. In cattle late embryo loss, while numerically much smaller than early embryo mortality loss, nevertheless, causes serious economic losses to producers because it is often too late to rebreed females when they repeat. In multiple ovulating small ruminants, the loss rate is positively related to ovulation rate. Systemic concentrations of progesterone, during both the cycle preceding and following insemination, affect embryo survival rate with evidence that too high or indeed too low a concentration being negatively associated with survival rate. Uterine expression of mRNA for progesterone receptor, oestradiol receptor and retinol-binding protein appears to be sensitive to changes in peripheral concentrations of progesterone during the first week after artificial insemination. Energy balance and dry matter intake during 4 weeks after calving are critically important in determining conception rate when cows are inseminated at 70-100 days post-calving. Concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates though sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy balance must be developed. There is sufficient genetic variability within the Holstein breed for fertility traits. Alternative dairy breeds such as the Jersey or Norwegian Red could also be utilized. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilization and the establishment of pregnancy, but also, in the future, identify genes responsible for improved embryo survival. Its incorporation into breeding objectives would increase the rate of genetic progress for embryo survival.

Negative energy balance alters global gene expression and immune responses in the uterus of postpartum dairy cows

Most dairy cows suffer uterine microbial contamination postpartum. Persistent endometritis often develops, associated with reduced fertility. We used a model of differential feeding and milking regimes to produce cows in differing negative energy balance status in early lactation (mild or severe, MNEB or SNEB). Blood hematology was assessed preslaughter at 2 wk postpartum. RNA expression in endometrial samples was compared using bovine Affymetrix arrays. Data were mapped using Ingenuity Pathway Analysis. Circulating concentrations of IGF-I remained lower in the SNEB group, whereas blood nonesterified fatty acid and β-hydroxybutyrate concentrations were raised. White blood cell count and lymphocyte number were reduced in SNEB cows. Array analysis of endometrial samples identified 274 differentially expressed probes representing 197 recognized genes between the energy balance groups. The main canonical pathways affected related to immunological and inflammatory disease and connective tissue disorders. Inflammatory response genes with major upregulation in SNEB cows included matrix metalloproteinases, chemokines, cytokines, and calgranulins. Expression of several interferon-inducible genes including ISG20, IFIH1, MX1, and MX2 were also significantly increased in the SNEB cows. These results provide evidence that cows in SNEB were still undergoing an active uterine inflammatory response 2 wk postpartum, whereas MNEB cows had more fully recovered from their energy deficit, with their endometrium reaching a more advanced stage of repair. SNEB may therefore prevent cows from mounting an effective immune response to the microbial challenge experienced after calving, prolonging the time required for uterine recovery and compromising subsequent fertility.

Embryo death in cattle: an update

For heifers, beef and moderate-yielding dairy cows, fertilisation generally exceeds 90%. In high-producing dairy cows, it may be lower and possibly more variable. The major component of embryo loss occurs before Day 16 following breeding, with emerging evidence of greater losses before Day 8 in high-producing dairy cows. Late embryo loss causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during the cycles both preceding and following insemination affect embryo survival; too-high or too-low a concentration has been shown to be negatively associated with survival rate. Energy balance and dry matter intake during the 4 weeks after calving are critically important in determining conception rate when cows are inseminated 70 to 100 days after calving. More balanced breeding strategies with greater emphasis on fertility, feed intake and energy must be developed. Genetic variability for fertility traits can be exploited; genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but could identify genes responsible for improved embryo survival. Their incorporation into breeding objectives would increase the rate of genetic progress for embryo survival. There is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial overall effect on herd reproductive performance.

Effect of progesterone on embryo survival

Increased genetic selection over the past 40 years has resulted in a dairy cow with an improved biological efficiency for producing milk but with an associated reduced fertility. Embryo loss is the greatest factor contributing to the failure of a cow to conceive. The extent and timing of embryo loss indicates that 70% to 80% of this loss occurs in the first 2 weeks after artificial insemination (AI). This is the period when a number of critical phases in embryo development occur and where protein accretion, substrate utilization and embryo metabolism increase dramatically. During this time the early embryo is completely dependent on the oviduct and uterine environment for its survival and it is likely that the embryo requires an optimal uterine environment to ensure normal growth and viability. There is increasing evidence of an association between the concentration of systemic progesterone and early embryo loss and that progesterone supplementation of cows, particularly those with low progesterone, can reduce this loss. While progesterone is known to affect uterine function and embryo growth, little is known about the uterus during the period of early embryo loss and how this is affected by changes in the concentration of systemic progesterone. The expression of uterine genes encoding the transport protein retinol binding protein (RBP) and the gene for folate binding protein (FBP) appear to be sensitive to changes in systemic progesterone, particularly during the early luteal phase of the cycle. Uterine concentrations of proteins also seem to be regulated by stage of cycle; however, their relationship with the systemic concentration of progesterone is unclear. There is an urgent need to characterize the uterine environment from a functional perspective during the early part of the luteal phase of the cycle, particularly in the high-producing cow, in order to understand the factors contributing to early embryo loss and in order to devise strategies to minimize or reduce this loss.

Effect of Elevated Systemic Concentrations of Ammonia and Urea on the Metabolite and Ionic Composition of Oviductal Fluid in Cattle

Abstract High dietary protein leads to elevated systemic concentrations of ammonia and urea, and these, in turn, have been associated with reduced fertility in cattle. The effect of elevating systemic concentrations of ammonia and urea on the concentrations of electrolytes and nonelectrolytes in bovine oviductal fluid were studied using estrus-synchronized, nulliparous heifers (n = 25). Heifers were randomly assigned to 1 of 3 treatments consisting of jugular vein infusion with either ammonium chloride (n = 8), urea (n = 8), or saline (n = 9). Oviducts were catheterized, and fluid was recovered over a 3-h period on either Day 2 or 8 of the estrous cycle. No difference (P > 0.05) was found in the concentrations of any electrolyte or nonelectrolyte between oviducts ipsi- or contralateral to the corpus luteum. Plasma and oviductal concentrations of urea were increased by infusion with urea (P < 0.001) and ammonium chloride (P < 0.05) but not by saline (P > 0.05). Plasma and oviductal concentrations of ammonia were elevated by infusion with ammonium chloride (P < 0.001) but not by infusion with urea or saline (P > 0.05). No effect (P > 0.05) of treatment was found on oviductal or plasma concentrations of glucose, lactate, magnesium, potassium, or sodium or on plasma concentrations of insulin or progesterone. The concentration of calcium in oviductal fluid was reduced by urea infusion and was negatively associated with systemic and oviductal concentrations of urea. Oviductal concentrations of sodium were higher on Day 8 than on Day 2 (P < 0.05). No effect of sample day was found on any of the other electrolytes or nonelectrolytes measured (P > 0.05). Elevated systemic concentrations of ammonia and urea are unlikely to reduce embryo survival through disruptions in the oviductal environment.

Effects of changes in the concentration of systemic progesterone on ions, amino acids and energy substrates in cattle oviduct and uterine fluid and blood

Early embryo loss is a major factor affecting the conception rate in cattle. Up to 40% of cattle embryos die within 3 weeks of fertilisation while they are nutritionally dependent on oviduct and uterine fluids for their survival. Inadequate systemic progesterone is one of the factors contributing to this loss. We have characterised the effects of changes in systemic progesterone on amino acid, ion and energy substrate composition of oviduct and uterine fluids on Days 3 and 6, respectively, of the oestrus cycle in cattle. Oviduct and uterine fluids were collected in situ following infusion of progesterone. There was no effect of progesterone on oviduct fluid secretion rate; however, uterine fluid secretion rate was lowered. Progesterone increased uterine glucose, decreased oviduct sulfate and, to a lesser degree, oviduct sodium, but had no effect on any of the ions in the uterus. The most marked effect of progesterone was on oviducal amino acid concentrations, with a twofold increase in glycine, whereas in the uterus only valine was increased. These results provide novel information on the maternal environment of the early cattle embryo and provide further evidence of progesterone regulation of oviduct amino acid concentrations in cattle.

Negative energy balance and hepatic gene expression patterns in high-yielding dairy cows during the early postpartum period: a global approach

In high-yielding dairy cows the liver undergoes extensive physiological and biochemical changes during the early postpartum period in an effort to re-establish metabolic homeostasis and to counteract the adverse effects of negative energy balance (NEB). These adaptations are likely to be mediated by significant alterations in hepatic gene expression. To gain new insights into these events an energy balance model was created using differential feeding and milking regimes to produce two groups of cows with either a mild (MNEB) or severe NEB (SNEB) status. Cows were slaughtered and liver tissues collected on days 6–7 of the first follicular wave postpartum. Using an Affymetrix 23k oligonucleotide bovine array to determine global gene expression in hepatic tissue of these cows, we found a total of 416 genes (189 up- and 227 downregulated) to be altered by SNEB. Network analysis using Ingenuity Pathway Analysis revealed that SNEB was associated with widespread changes in gene expression classified into 36 gene networks including those associated with lipid metabolism, connective tissue development and function, cell signaling, cell cycle, and metabolic diseases, the three most significant of which are discussed in detail. SNEB cows displayed reduced expression of transcription activators and signal transducers that regulate the expression of genes and gene networks associated with cell signaling and tissue repair. These alterations are linked with increased expression of abnormal cell cycle and cellular proliferation associated pathways. This study provides new information and insights on the effect of SNEB on gene expression in high-yielding Holstein Friesian dairy cows in the early postpartum period.

Pleiotropic effects of negative energy balance in the postpartum dairy cow on splenic gene expression: repercussions for innate and adaptive immunity

Increased energy demands to support lactation, coupled with lowered feed intake capacity results in negative energy balance (NEB) and is typically characterized by extensive mobilization of body energy reserves in the early postpartum dairy cow. The catabolism of stored lipid leads to an increase in the systemic concentrations of nonesterified fatty acids (NEFA) and β-hydroxy butyrate (BHB). Oxidation of NEFA in the liver result in the increased production of reactive oxygen species and the onset of oxidative stress and can lead to disruption of normal metabolism and physiology. The immune system is depressed in the peripartum period and early lactation and dairy cows are therefore more vulnerable to bacterial infections causing mastitis and or endometritis at this time. A bovine Affymetrix oligonucleotide array was used to determine global gene expression in the spleen of dairy cows in the early postpartum period. Spleen tissue was removed post mortem from five severe NEB (SNEB) and five medium NEB (MNEB) cows 15 days postpartum. SNEB increased systemic concentrations of NEFA and BHB, and white blood cell and lymphocyte numbers were decreased in SNEB animals. A total of 545 genes were altered by SNEB. Network analysis using Ingenuity Pathway Analysis revealed that SNEB was associated with NRF2-mediated oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, natural killer cell signaling, p53 signaling, downregulation of IL-15, BCL-2, and IFN-γ; upregulation of BAX and CHOP and increased apoptosis with a potential negative impact on innate and adaptive immunity.

Effect of systemic progesterone concentration on the expression of progesterone-responsive genes in the bovine endometrium during the early luteal phase

Increasing evidence indicates an association between the concentration of systemic progesterone during the early luteal phase of the oestrous cycle and embryo survival rate in cattle. We examined the relationship between the concentration of systemic progesterone on Days 4 to 8 post-ovulation and expression of progesterone receptor (PGR), oestrogen receptor +/- (ESR1) and retinol-binding protein (RBP) mRNA in the bovine endometrium. Heifers were blood sampled from the day of ovulation (Day 0) to Day 8 post-ovulation. On Day 4, animals were divided into low progesterone control (LC) and high progesterone control (HC) groups based on their plasma progesterone concentrations. Half of each group was supplemented with exogenous progesterone resulting in two further groups, low progesterone supplemented (LS) and high progesterone supplemented (HS). Endometrial tissues were recovered from all groups on Day 6 or Day 8 and gene expression was analysed following Northern blotting. Increasing progesterone concentrations were associated with decreased PGR and ESR1 expression. Duration-dependent effects of progesterone supplementation on ESR1 were evident and there was an effect of systemic progesterone concentrations between Day 0 and Day 4 on the expression of RBP at Days 6 and 8. Such progesterone-responsive changes in uterine gene expression are likely to affect embryo development.

Postmortem stability of RNA isolated from bovine reproductive tissues

Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.