Energy balance and size and number of ovarian follicles detected by ultrasonography in early postpartum dairy cows

Multiparous Holstein cows (n = 52) were fed one of six diets consisting of a totally mixed ration (corn silage, corn grain, soybean meal, dried distillers grains, and whole cottonseed) plus either alfalfa hay, alfalfa cubes, or bermuda-grass hay fed chopped as a component in the mixed ration or separate as long hay. Predicted energy balance was calculated from DM intake, milk yield and composition, and BW. On d 25 postpartum, ovarian status was programmed by injecting 25 mg of prostaglandin F2 alpha and treating cows for 15 d with an intravaginal device containing 1.9 g progesterone. Before d 25, number of class 1 follicles (3 to 5 mm; detected by ultrasonography) decreased with increasing days postpartum, and number of class 3 (10 to 15 mm) and class 4 (greater than 15 mm) follicles increased. The number of class 1 and 2 follicles (6 to 9 mm) decreased with increasing energy balance, and number of class 3 follicles increased with energy balance. Before d 25, predicted energy balance explained treatment differences in the number of follicles within each size class. After d 25, energy balance did not affect the average number of follicles per cow, but diet affected the number of follicles within each class. Predicted energy balance and dietary treatments influenced number of follicles at different times after calving. These results identify the importance of diet and energy balance to follicular and ovarian function in postpartum lactating dairy cows.     

Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle

First postpartum dominant follicles are preferentially selected in the ovary contralateral to the previously gravid uterine horn. The aim of the present study was to test the hypothesis that uterine bacterial contamination alters the location of ovarian follicle emergence and selection, and inhibits follicle growth and function. Swabs were collected from the uterine body lumen of cattle on days 7, 14, 21 and 28 after parturition. Bacteria were identified by aerobic and anaerobic culture; bacterial growth was scored semiquantitatively and animals were categorized into standard or high bacterial contamination categories on the basis of the number of colonies detected. Follicular growth and function were monitored by daily transrectal ultrasonography, and estimation of plasma FSH, oestradiol and progesterone concentrations. There was no effect of bacterial contamination on plasma FSH concentration profiles or emergence of the ovarian follicle wave. When uterine bacterial growth scores were high on day 7 or day 21 after parturition, fewer first (1/20 versus 15/50; P < 0.05) or second (1/11 versus 13/32; P < 0.05) dominant follicles were selected in the ipsilateral compared with the contralateral ovary, respectively. The diameter of the first dominant follicle was smaller in animals with a high day 7 bacterial score (P < 0.001), dominant follicle growth was slower (P < 0.05) and oestradiol secretion was decreased (P < 0.05). The present study provides evidence for an effect of the uterus on the ovary after parturition, whereby uterine bacteria have a contemporaneous localized effect on ovarian follicle selection and subsequent growth and function, but not on initial emergence.     

Role of peripartum dietary propylene glycol or protected fats on metabolism and early postpartum ovarian follicles

Eighty multiparous cows were used to test the effects of feeding a supplement containing 55% dry propylene glycol (PGLY), prilled fat (PrFA) containing a low proportion of unsaturated fatty acids (FA), or calcium soaps of long-chain FA (CaLFA) containing a high proportion of unsaturated FA on energy balance (EB), blood metabolites, and early postpartum (PP) ovarian follicles. Dry cows (256 d pregnant) were divided into 6 groups and began the following dietary treatments: 1) control group, fed a dry cow diet and fed a lactating cow diet PP; 2) PGLY group, diet supplemented with 500 g/d per cow of dry PGLY prepartum through 21 d in milk; 3) PrFA:control group, diet supplemented with 230 g/d per cow of PrFA prepartum and fed the control diet PP; 4) PrFA:PrFA group, diet supplemented with 230 g/d per cow of PrFA prepartum through 21 d in milk; 5) CaLFA:control, supplemented with 215 g/d per cow of CaLFA prepartum and fed the control diet PP; 6) CaLFA:CaLFA, supplemented with 215 g/d per cow of CaLFA prepartum through 21 d in milk. Follicular fluid was aspirated from follicles ≥6 mm on d 12 PP. The daily average calculated EB during the first 21 d in milk was lower in the PrFA:PrFA (−4.16 Mcal/d) and CaLFA:CaLFA (−3.64 Mcal/d) groups than in the control (−1.71 Mcal/d) and PGLY (−2.19 Mcal/d) groups. Postpartum plasma β-hydroxybutyrate was higher, and insulin concentrations were lower in the PrFA:PrFA (6.2 mg/dL and 126.1 pg/mL, respectively) and CaLFA:CaLFA (7.0 mg/dL and 130.7 pg/mL) groups than in the control (4.5 mg/dL and 274.5 pg/mL) and PGLY (4.3 mg/dL and 272.6 pg/mL) groups, whereas nonesterified FA concentrations were higher only than the control group. Postpartum nonesterified FA were 21% lower and insulin plasma concentrations were 86% higher in the CaLFA:control group as compared with the PrFA:control group. The progesterone concentrations in the follicular fluid of estradiol-active follicles were higher in the CaLFA:CaLFA (200.7 ng/mL) group than in all other groups (57.3 to 92.4 ng/mL), and androstenedione and estradiol concentrations were higher (54.2 and 1,049.1 ng/mL, respectively) than in the PGLY (15.5 and 440.1 ng/mL), PrFA:control (22.6 and 314.1 ng/mL), and CaLFA:control (17.5 and 451.9 ng/mL) groups. In conclusion, supplementation of protected fat during the peripartum period negatively affected the EB status of the cows. Neither fat supplementation nor PGLY influenced the development of ovarian follicles during the early PP period, but feeding fat containing a high ratio of unsaturated FA (CaLFA) increased progesterone concentrations in estradiol-active follicles that were aspirated at 12 d in milk.     

Identification of potential markers in blood for the development of subclinical and clinical mastitis in dairy cattle at parturition and during early lactation

Our objective was to identify specific blood markers as risk factors for the development of mastitis during early lactation. We used a subset of cows from a larger experiment that consisted of a total of 634 lactations from 317 cows. Cows were of 3 breeds and ranged from parity 1 to 4. Blood samples were collected weekly from 56 d before expected calving date through 90 d in milk (DIM). Blood was analyzed for several hormones, metabolites, and enzymes, and energy intake and energy balance were calculated. Veterinary treatment records and daily composite milk somatic cell counts were analyzed and used to determine incidence and severity of mastitis in early lactation. Cows were separated into 2 groups: 1) WK0, consisting of cows that developed clinical mastitis (CM), cows that developed subclinical mastitis (SM), or cows that were healthy (H) during the first 7 DIM; and 2) EL, consisting of CM, SM, or H cows during wk 2 through 13 of lactation. Data were adjusted for numerous fixed effects (e.g., parity, breed, season, and DIM) before statistical analysis. The time of mastitis (TOM) was recorded as the DIM in which the first rise in somatic cell count was observed and was recorded as TOM = 0. The time before and after TOM was distinguished as ± n wk relative to TOM = 0. Healthy cows were paired with either a SM or CM cow and the TOM for each H cow was equal to the TOM for its paired SM or CM cow. Data from wk −1 and −2 relative to TOM were analyzed for group WK0 and EL, respectively. For all parameters, SM cows did not differ from H cows from either group. The CM cows had higher nonesterified fatty acid levels and a tendency toward higher β-hydroxybutyrate levels than H cows before mastitis for both groups. For group WK0, glucose was higher −1 wk relative to calving in CM than H cows. For group EL, aspartate aminotransferase was higher −2 wk relative to mastitis in CM than H cows during 8 to 90 DIM. All other variables were similar among CM, SM, and H cows for both groups. Our results indicate that substances in blood, especially nonesterified fatty acids and aspartate aminotransferase, may be potential markers for the risk of mastitis in early lactation.     

Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows

Ovarian follicular cysts and persistent follicles are follicular pathologies involved in reduced fertility of dairy cows. Two separate experiments were performed on high-yielding Holstein cows to characterize ovarian cyclicity and evaluate the developmental dynamics of follicle pathologies postpartum. In experiment 1, 58 cows were monitored by ultrasonography twice weekly from d 18±1 to 69±2 postpartum. First ovulation occurred 38±3, 27±2, 20±1, and 25±3 d postpartum in cows with 1 cycle (n=11), 2 cycles (n=21), 3 cycles (n=13), and 4 cycles (n=7), respectively. Follicular pathologies were developed in cows that were either acyclic (n=6) or had 1 or 2 cycles, but not in cows with more than 2 cycles. In experiment 2, 47 cows were monitored twice weekly from 10 d postpartum to second ovulation. Follicles ≥17 mm in diameter in 2 consecutive scans were aspirated, and concentrations of various hormones were measured. Cows were defined as cyclic (n=30; 64%) or with the potential to develop follicular pathology (n=17; 36%). Aspirated follicles (n=27) were classified into 3 main groups based on follicular growth rate, follicular diameter, and ovarian activity before and after follicular aspiration. Dominant follicles (n=4) were defined as large follicles (20 mm in diameter) with growth rate ≤1 mm/d and normal ovarian activity. Persistent follicles (n=6) had the same growth rate and diameter as the dominant follicles, but persisted at the same diameter for ≥10 d. Ovarian cysts (n=17) were defined as the largest follicular structures (19 to 32 mm in diameter), with abnormal growth rate (>1 mm/d) and abnormal ovarian activity. Single or turnover cysts did not differ in their growth parameters and were therefore combined and further classified according to follicular-fluid hormone concentrations. Estradiol-dominant cysts (n=7) were characterized by normal estradiol (284 to 659 ng/mL) and progesterone (20 to 113 ng/mL) concentrations, similar to those of the dominant follicle (554 to 993 ng/mL and 44 to 106 ng/mL, respectively). Progesterone-dominant cysts (n=5) were characterized by low estradiol (0.06 to 330 ng/mL) and high progesterone (586 to 3,288 ng/mL) concentrations. Low-steroidogenic active cysts (n=5) were characterized by low concentrations of both estradiol (23 to 61 ng/mL) and progesterone (17 to 205 ng/mL). Characterization of spontaneously forming cysts might enable definition of the formation of ovarian follicular pathologies in postpartum cows.     

Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro

Ascorbic acid has three known functions: it is necessary for collagen synthesis, promotes steroidogenesis and acts as an antioxidant. Within the ovary, most studies have concentrated on the role of ascorbic acid in luteal formation and regression and little is known about the function of this vitamin in follicular growth and development. Follicular growth and development were investigated in this study using an individual follicle culture system that allows the growth of follicles from the late preantral stage to Graafian morphology. Follicles were isolated from prepubertal mice and cultured for 6 days. Control media contained serum and human recombinant FSH. Further groups of follicles were cultured in the same media but with the addition of ascorbic acid at concentrations of either 28 or 280 micromol l(-1). Addition of ascorbic acid at the higher concentration significantly increased the percentage of follicles that maintained basement membrane integrity throughout culture (P < 0.001). Ascorbic acid had no effect on the growth of the follicles or on oestradiol production. Metalloproteinase 2 activity tended to increase at the higher concentration of ascorbic acid and there was a significant concomitant increase in the activity of tissue inhibitor of metalloproteinase 1 (P < 0.01). Follicles cultured without the addition of serum but with FSH and selenium in the culture media underwent apoptosis. Addition of ascorbic acid to follicles cultured under serum-free conditions significantly reduced apoptosis (P < 0.05). From these data it is concluded that ascorbic acid is necessary for remodelling the basement membrane during follicular growth and that the ability of follicles to uptake ascorbic acid confers an advantage in terms of granulosa cell survival.     

Regulation of ovarian follicular growth by somatotropin and insulin-like growth factors in cattle

Somatotropin (ST), insulin-like growth factor (IGF)-I, and IGF-II affect animal growth and lactation as well as animal reproduction. Understanding the effects of ST and the IGF on reproduction is important because ST and IGF-I undergo dynamic changes prior to the postpartum breeding period. In addition, administration of recombinant bovine somatotropin (rbST) to lactating cows is a common practice that increases blood concentrations of ST and IGF-I during the breeding period. In vivo, administration of rbST caused greater ovarian follicular development. The effects of rbST may represent direct actions of ST because ST receptors are found within granulosa cells as well as oocytes. Alternatively, the actions of ST may be indirectly mediated by increased IGF-I and (or) nutrient partitioning that occurs after rbST. Both IGF-I and IGF-II are synthesized within the ovary. Ovarian IGF are, therefore, a composite of IGF from both endocrine (liver) and autocrine and paracrine (ovary) sources. The IGF stimulate ovarian function by acting synergistically with gonadotropins to promote growth and steroidogenesis of ovarian cells. Actions of IGF-I and -II are restrained by a series of IGF binding proteins (IGFBP) that either originate from the blood or are synthesized locally within the follicle. Degradation and differential synthesis of IGFBP are important mechanisms regulating IGFBP amounts. The relative amounts of IGFBP may ultimately determine ovarian IGF action. Future studies of ST and IGFs should focus on the hormones, receptors, and binding proteins as well as the metabolic requirements for normal ovarian function in dairy cattle.     

Effects of whole cottonseed diet and recombinant bovine somatotropin on ovarian follicles in lactating dairy cows

The effects of whole cottonseed (WCS) in the diet and the administration of bovine somatotropin (bST) on ovarian follicular dynamics and plasma progesterone (P4) concentrations were examined in cows during a period of synchronized follicular growth. Lactating Holstein cows (n = 28) were randomly assigned to treatments in a 2 x 2 factorial arrangement. Diets consisted of WCS (15% of dry matter) or no WCS, and bST at a dose of 0 or 208 mg/14 d. Dietary treatments began within 24 h of calving and bST treatments began within 7 d postpartum. Cows received GnRH at 65 +/- 3 d postpartum (d 0), PGF2alpha, (d 7), a second dose of GnRH (d 9), and were inseminated 16 h later (d 10). Ovarian changes were monitored daily by ultrasonography from d 0 to 9. On d 9,93% of cows had a preovulatory follicle and 86% ovulated. For Class 2 (6 to 9 mm) follicles, a diet x bST interaction was detected, with bST stimulating Class 2 follicles in cows fed WCS, but not in cows on the control diet. Neither diet nor bST affected numbers of Class 1 (2 to 5 mm) or Class 3 (> or = 10 mm) follicles or sizes of the subordinate and dominant follicles. During the luteal phase of the cycle, lactating cows fed WCS tended to have elevated concentrations of plasma P4, whereas bST was without effect. Plasma concentrations of high-density lipoprotein cholesterol were increased in cows fed WCS. Number and diameter of corpora lutea did not differ among treatments.     

Comparative study of metabolic differentiation of foetal muscle in normal and double-muscled cattle

Studies of the contractile differentiation of foetal muscles in normal and double-muscled cattle have shown that double-muscled foetal muscles display comparative delayed contractile differentiation. We compared the metabolic differentiation of foetal muscles in normal and double-muscled cattle, for which no data were available. The semitendinosus (ST) muscle was excised from normal and double-muscled cattle foetuses aged 110-260 days. Isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities, respectively representative of aerobic oxidative and anaerobic glycolytic metabolism, were assayed. The five LDH isozymes were separated by electrophoresis under non-denaturing conditions and assayed by densitometry. ICDH activity was weaker in the ST of double-muscled foetuses than in that of normal ones throughout gestation, and it increased later in the double-muscled foetuses than in normal ones. In contrast, the LDH activity increased at 180 days in both. It was greater in the double-muscled beyond that time. The proportion of LDH-M subunit was higher at all foetal ages in the double-muscled. Overall, these results show that: (1) the metabolic differentiation of muscle fibre occurs during the last third of foetal growth in both normal and double-muscled cattle; and (2) the differentiation of aerobic oxidative metabolism is delayed in double-muscled foetuses compared with normal ones.     

Influence of concentrate allocation and energy balance on postpartum ovarian activity in Norwegian cattle

The effects of different levels of concentrate, milk yield, and energy balance (EB) on postpartum ovarian activity were studied in 164 lactations from 108 moderate yielding, dual-purpose cows. All cows were of the breed Norwegian Cattle. The cows were assigned to three different concentrate allocations and had free access to roughage. Cows in the high energy group were fed as suggested by Norwegian standards, whereas cows in the low energy group were supplied with approximately 40% less concentrate than recommended. Cows in the medium energy group were fed as the low energy group prepartum and as the high energy group during the first 90 d in milk (DIM). Energy balance was estimated by subtracting energy required for maintenance and lactation from energy intake. Ovarian activity was expressed in categories (early, mid and late responders) of days to first luteal response (milk progesterone concentration >5 ng/ml) and days to first regular cycle. Energy balance and milk yield throughout the first 100 DIM were compared between categories of concentrate allocation and ovarian activity using repeated measures mixed models. Decreasing level of concentrate was related to negative EB and declining milk yield. Both negative EB and high milk yield were associated with delayed postpartum ovarian activity. Concentrate allocation did not, however, alter days to first luteal response or days to first cycle. This study demonstrated that although negative EB was associated with delayed ovulation, Norwegian Cattle adapted to less energy-dense rations by reducing milk yield rather than delaying onset of ovarian activity.     

Relationships between energy balance and health traits of dairy cattle in early lactation

The objective of the study was to calculate phenotypic relationships between energy balance in early lactation and health and reproduction in that lactation. Data were 26,701 daily records of dry matter intake and milk production, periodic measures of milk composition and body weight, and all health and reproductive information from 140 multiparous Holstein cows. Daily energy balance was calculated by multiplying feed intake by the concentration of energy of the ration and subtracting the amount of energy required for maintenance (based on parity and body weight) and for milk production (based on yield and concentrations of fat, protein, and lactose). Six measures of energy balance were defined: mean daily energy balance during the first 20, 50, and 100 d of lactation; minimum daily energy balance; days in negative energy balance; and total energy deficit. Measures of health were the numbers of occurrences of each of the following during lactation: all udder problems, mastitis, all locomotive problems, laminitis, digestive problems, and reproductive problems. Reproductive traits were the number of days to first observed estrus and number of inseminations. Several significant relationships between energy balance and health were observed. Increased digestive and locomotive problems were associated with longer and more extreme periods of negative energy balance.     

Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis

Growth differentiation factor-9 (GDF-9) is a growth factor secreted by oocytes in growing ovarian follicles. To investigate the ovarian function of GDF-9 in pigs, we first cloned porcine GDF-9 complementary DNA (cDNA), and then injected its gene fragments into the ovary in gilts. Porcine GDF-9 has open reading frame (ORF) homologies of 81.4%, 84.6%, 84.2%, 72.7% and 72.6% with its human, bovine, ovine, rat and mouse counterparts respectively. Regarding the deduced amino-acid sequence of the mature protein, the corresponding homologies reach 92.1%, 97.8%, 97.0%, 89.6% and 88.1% respectively. To investigate the role of GDF-9 in early folliculogenesis, the ovaries of 2-month-old prepubertal gilts were injected with GDF-9 gene fragments. The injection of porcine GDF-9 gene fragments resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles. These results indicated that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary, and that a technique for direct ovarian injection of GFD-9 gene fragments may contribute to a novel therapy for prevention and treatment of infertility associated with ovarian dysfunction.     

Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.     

Extruded blend of soybean meal and sunflower seeds for dairy cattle in early lactation

An extruded blend of 44% crude protein soybean meal (50%), whole sunflower seeds (45%), and premix (5%) was evaluated as a protein and energy source for dairy cows in early lactation. Thirty Holstein cows (24 multiparous and 6 primiparous) were assigned to either a corn-oats-soybean meal concentrate or a concentrate where soy-sunflower blend replaced all soybean meal and portions of corn and oats. Dry matter of total mixed diets was 36% corn silage, 21% alfalfa haylage, and 43% concentrate. Yields of milk (33.6 and 33.8 kg/d) and 4% fat-corrected milk (30.9 and 30.5 kg/d) were similar, and percentages of total solids (11.92 and 11.38), fat (3.55 and 3.30), and protein (2.91 and 2.74) were lower in milk from cows fed soy-sunflower blend. Milk from cows fed soy-sunflower blend contained fewer short- and medium-chain fatty acids, more 18-carbon fatty acids, and was more unsaturated than from cows fed soybean meal. Intakes of dry matter and changes in body weight were not different among diets. Ruminal fluid pH and molar ratio of acetate to propionate were higher, and concentrations of total volatile fatty acids and ammonia were lower in cows fed soy-sunflower blend. Concentrations of essential amino acids in arterial serum, calculated mammary uptakes, and transfer efficiencies indicated more desirable amino acid balance in cows fed soy-sunflower blend. Methionine appeared to be most limiting with both diets.     

Evaluation of biochemical parameters and genetic markers for association with meat tenderness in South African feedlot cattle

A large proportion of South African feedlot cattle are crossbreds of Brahman (BrX, Bos indicus), and Simmental (SiX, Bos taurus). A sample of 20 grain fed bulls from each of these crossbreeds was used to compare meat quality with that of the small frame indigenous Nguni (NgX, Sanga) by evaluating a variety of biochemical and genetic parameters previously shown to be associated with meat tenderness. Shear force values were generally high (5.6 kg average at 14 days post mortem), with SiX animals higher than BrX or NgX (P = 0.051) despite higher calpastatin:calpain ratio in BrX (P < 0.05). Calpain activity and cold shortening were both correlated with tenderness for all classes. The sample size was too small to accurately estimate genotypic effects of previously published markers in the CAST and CAPN1 genes, but the allele frequencies suggest that only modest progress would be possible in these South African crossbreds using these markers.     

Detection of QTL for milk protein percentage in Italian Friesian cattle by AFLP markers and selective genotyping

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on six Bos taurus (BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.     

Implication of cortisol and 11beta-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts

This study investigated cortisol inactivation by 11beta-hydroxysteroid dehydrogenase (11beta HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP(+)-dependent 11beta-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11beta HSD, this studyalso investigated whether levels of 11beta HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11beta HSD1 activity in rat kidney homogenates decreased progressively from 50 +/- 8% inhibition by FF from small antral follicles (P < 0.001) to 23 +/- 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11beta HSD1 activity by 59 +/- 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35-48% inhibition, P < 0.05) and cyst fluid (45-75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11beta HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11beta HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.