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.     

Effect of calcium soaps of fatty acids and administration of somatotropin on milk production, preovulatory follicular development, and plasma and follicular fluid lipid composition in high yielding dairy cows

The effect of fat and bovine somatotropin (bST) on preovulatory follicular hormones and lipids was evaluated by feeding cows for 150 d from parturition a control diet, a control diet plus 0.55 kg/d of calcium soaps of fatty acids, or a control diet with 500 mg of bST injected every 14 d. Fourteen days after a synchronized or natural estrus, cows were injected with a PGF2 alpha analogue; 48 h later, follicular fluid from all ovarian follicles > 8 mm was aspirated. Cows fed fat or injected with bST produced more milk and milk solids than did control cows, and cows on the bST treatment lost more body condition after calving than did cows on the other treatments. Both treatments changed the proportion of estradiol-active follicles (> 400 ng of estradiol/ml of follicular fluid) and the correlation between follicular fluid estradiol concentration and the total number large follicles per cow. In follicles aspirated between 60 and 90 DIM the percentage of estradiol-active follicles was 67, 40, and 0 for cows on the control, calcium soaps of fatty acids, and bST treatments, respectively. After 90 DIM, no differences existed between treatments in the percentage of estradiol-active follicles. Estradiol concentration in follicular fluid was correlated with DIM at follicle aspiration (r = 0.51). The proportion of oleic acid in free fatty acids in plasma at 50 DIM was lower in control cows and was lower in follicular fluid of estradiol-active follicles. Both calcium soaps of fatty acids and bST had a considerable effect on follicular development and activity and the composition of fatty acids in follicles.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Induction of persistent ovarian follicular structures following administration of progesterone near the onset of estrus in dairy cattle

The objective of this study was to determine if progesterone administered near the onset of estrus in dairy cows would block the preovulatory surge of LH and result in subsequent persistence of ovarian follicular structures. Following synchronization of estrus with prostaglandin F2 alpha, 20 multiparous, non-lactating Holstein cows were randomly assigned into three groups: 1 ml ethanol administered i.m. at 12-h intervals for 24 h (n = 6; group 1); 1 mg progesterone administered i.m. at 12-h intervals for 24 h (n = 7; group 2); 2.5 mg progesterone administered i.v. at the onset of standing estrus (n = 7; group 3). Ovarian structures were palpated per rectum on the day of estrus and twice weekly for 14 d. Blood was collected every 2 h from onset of standing estrus for 30 h, and concentrations of LH and progesterone were determined. Numbers of cows diagnosed with persistent follicles 10 d after estrus were 1 of 6 (group 1), 2 of 7 (group 2), and 5 of 7 (group 3). The preovulatory surge of LH did not occur during the sampling period (30 h) for 1 of 6, 7 of 7 and 5 of 7 cows, for groups 1, 2, and 3, respectively, and mean serum concentrations of LH were higher in group 1 than in groups 2 and 3. Serum concentration of progesterone (ng/ml) was higher in group 1 (1.9 +/- .4) than in groups 2 (.9 +/- .4) and 3 (.9 +/- .4) 10 d following estrus. Blocking the preovulatory surge of LH with exogenous progesterone resulted in persistence of ovarian follicles.     

Preovulatory follicle characteristics and oocyte competence in repeat breeder dairy cows

The varied and elusive etiology of repeat breeding (RB) in dairy cows necessitates evaluation of oocytes and follicles, which have not previously been assessed together. Accordingly, we evaluated characteristics of preovulatory follicles and the competence of oocytes in control (CTL) and RB cows. The estrous cycles of 35 cows (18 CTL and 17 RB) were synchronized using PGF_2α and estrus detection. Cows with a corpus luteum were treated with PGF_2α and, 14 to 15 d after a visible behavioral estrus, they were administered a second PGF_2α, followed 48 h later by follicular fluid (FF) aspiration of the preovulatory follicles. Estradiol (E₂)-active preovulatory follicles did not differ in diameter between the 2 groups of cows. However, FF of RB cows had higher E₂ concentrations than that of CTL cows: 1,854.9 and 1,073.6 ng/mL, respectively, but similar androstenedione and progesterone concentrations. In the second part of the study, 14 consecutive ovum pick-ups (OPU) were performed in 5 CTL and 5 RB cows, at 3- to 4-d intervals. The RB and CTL cows did not differ in average numbers of follicles available per cow per session (7.1 and 7.3, respectively), oocyte recovery rates (42.2 and 44.1%, respectively), or cleavage rates (57.6 and 63.4%, respectively), but blastocyst production was markedly less in RB than in CTL cows (12.5 and 29.2%, respectively). We conclude that part of the RB cows' etiology occurs at an earlier phase of folliculogenesis, thereby impairing oocyte competence, and subsequently reducing the probability of normal fertilization, which diminish embryo vitality and development.     

Effects of dietary fats differing in n-6:n-3 ratio fed to high-yielding dairy cows on fatty acid composition of ovarian compartments, follicular status, and oocyte quality

The objectives were to determine the incorporation of dietary encapsulated fats differing in n-6:n-3 ratio into milk fat, plasma, and various ovarian compartments and to examine the effects on ovarian follicular status, preovulatory follicle characteristics, and oocyte quality. Twenty-four multiparous Israeli Holstein cows, averaging 114 d in milk, were assigned to 1 of 3 treatment groups: 1) control (n = 7), in which cows were fed a lactating cow diet; 2) E-FLAX (n = 8), in which cows were fed a lactating cow diet that consisted of 1 kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% flaxseed oil, providing 242.2 g of C18:3n-3 (low n-6:n-3 ratio); or 3) E-SUN (n = 9), in which cows were fed a lactating cow diet that consisted of 1 kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% sunflower oil, providing 260.0 g of C18:2n-6 (high n-6:n-3 ratio). Ovaries were monitored by ultrasonography for follicular status, and after synchronization, follicles >7 mm were aspirated and evaluated. Ovum pickup was performed (19 sessions for the control and E-FLAX groups and 11 for the E-SUN group), and in vitro maturation and oocyte fertilization were conducted. The E-FLAX treatment increased the proportions of C18:3n-3 (5.8 fold), C20:5n-3, and C22:5n-3 (approximately 4-fold) in milk fat as compared with the other 2 treatments. The proportion of C18:3n-3 fatty acid in plasma increased dramatically with the E-FLAX treatment, from 1.43 and 1.49% in the control and E-SUN groups, respectively, to 7.98% in the E-FLAX group. Consequently, the n-6:n-3 ratio in plasma was reduced from approximately 42 in the control and E-SUN groups to 6.74 in the E-FLAX group. Proportions of C18:3n-3 in follicular fluid and granulosa cells were approximately 5-fold higher in the E-FLAX group than in the other 2 groups. The percentage of C18:2n-6 in cumulus-oocyte complexes of cows in the E-SUN group was 54% higher than that in the E-FLAX group and was 2.4-fold higher than that in the control group; the proportion of C18:3n-3 in the E-FLAX group was 4.73% and was not detected in the other groups. The average numbers of 2- to 5-mm follicles on d 5 and 9 of the cycle were higher in the E-FLAX group than in the E-SUN group, whereas the average numbers of follicles ≥10 mm on d 5, 9, and 13 were higher in the E-SUN group than in the other 2 groups. The estrous cycles of the cows were synchronized and PGF_2α was injected on d 16 to 17 of the cycle. The interval from PGF_2α injection to behavioral estrus was longer in the E-FLAX group than in the E-SUN group, and the beginning of the luteal phase of the subsequent cycle was delayed. Concentrations of estradiol in follicular fluid of the preovulatory follicles were higher in the E-SUN group than in the E-FLAX group. The number of follicles aspirated by ovum pickup was higher in the E-FLAX group than in the control group, and the cleavage rate in the E-FLAX group was higher than in the control group, but not the E-SUN group. In conclusion, dietary n-3 fatty acids influenced the follicular status and increased the cleavage rate of oocytes as compared with those of control cows. These findings could be related to modifications of the fatty acid composition in plasma and ovarian compartments in response to dietary supplementation.     

Incorporation of dietary n-3 fatty acids into ovarian compartments in dairy cows and the effects on hormonal and behavioral patterns around estrus

The objective of this study was to examine the incorporation of dietary n-3 fatty acids (FAs) into ovarian compartments and the effects on hormonal and behavioral patterns around estrus. Multiparous 256-day pregnant cows were fed either a standard diet both prepartum and postpartum (PP) (control; n=22) or supplemented with extruded flaxseed (E-FLAX) providing C18:3n-3 at 172.2 and 402.5 g/day per cow prepartum and PP respectively (n=22). The estrous cycle was synchronized, and at day 7 of the cycle, the cows were injected with prostaglandin F(2)(α) (PGF(2)(α)) and then subjected to 5 days of intensive examination. Compared with those in the control, in the E-FLAX group, the interval from PGF(2)(α) injection to behavioral estrus peak tended to be longer (3.6 h; P<0.1), that to estradiol (E(2)) peak was 6.5 h longer (P<0.03), and that to LH peak tended to be longer (5.3 h; P<0.07). The durations of behavioral estrus and E(2) surge were longer, and the area under the E(2) curve was greater in the E-FLAX cows. Afterward, 7-8 days following behavioral estrus, follicular fluids (FFs) from >7 mm follicles were aspirated. The proportions of n-3 FA increased in plasma, FF, and granulosa cells in the E-FLAX group. The concentrations of PGE(2) in the E(2)-active follicles tended to be lower in the E-FLAX cows (P<0.06). In conclusion, several modifications in hormonal and behavioral estrus patterns were demonstrated in cows fed n-3 FA, which might be attributed to alterations in membrane FA composition and partly mediated by lower PGE(2) synthesis.     

Effects of the persistent dominant follicle on the ability of follicle stimulating hormone to induce follicle development and ovulatory responses

Three experiments were conducted to evaluate the effect of an induced first wave persistent dominant follicle on folliculogenesis and ovulatory responses induced by FSH. On d 6 of a synchronized estrous cycle (d 0 = estrus), cows were treated with a Syncromate-B implant and two injections of PGF2, (25 mg, 0700 h; 15 mg, 1900 h, i.m.). Cows in the control group retained a first-wave persistent dominant follicle, but in the aspirated group, the first-wave dominant follicle was removed via transvaginal aspiration on d 10 (d 0 = estrus). Beginning on d 12, cows received 32 mg of FSH-P i.m. in decreasing doses at 12-h intervals over a 4-d period. On d 15, the Syncromate-B implant was removed, and cows were ovariectomized (experiment 1, n = 8) or inseminated (experiment 2, n = 11) at 10 and 22 h after the onset of estrus. Cows in experiment 3 received a used controlled intravaginal drug releasing (CIDR) device and two injections of PGF2alpha (25 mg, 0700 h; 15 mg, 1900 h; i.m.) on d 6. On d 8, the first-wave dominant follicle was aspirated (n = 6) or left intact (n = 5), and FSH treatment was initiated (20 mg of Folltropin in decreasing doses at 12-h intervals over a 4-d period), and on d 10 the used CIDR device was removed from all cows. Ovarian follicle size and number were examined daily by ultrasonography from d 5 of the estrous cycle. The persistent dominant follicle increased in size from 10.7 mm on d 5 to 15.4 mm on d 10 (experiments 1 and 2), and from 9 mm on d 5 to 20.4 mm on d 11 (experiment 3). From d 11 to 14, the number of class 1 (2 to 5 mm) follicles was lower in the aspirated group than in the control group; the number of class 2 (6 to 9 mm) follicles was higher on d 12 and 13 for the aspirated group (experiments 1 and 2). The number of class 3 (> or =10 mm) follicles was higher in the aspirated group on d 14 to 16, but the same on d 17. Ovarian and embryo responses to superovulation did not differ between groups. In experiment 3, the numbers of class 1, 2, and 3 follicles, as well as ovarian and embryo responses following ovulation did not differ between groups. Initiation of exogenous FSH treatment appears to override any systemic inhibitory effect that a persistent dominant follicle may be exerting at the pituitary and possibly the ovary.     

Effects of high dietary crude protein on the characteristics of preovulatory follicles in dairy heifers

The objectives were to examine the effect of high dietary crude protein on characteristics of preovulatory follicles in dairy heifers. Eight Israeli-Holstein heifers, 4 fitted with rumen fistula and 4 intact, were assigned to 1 of 3 treatments in a replicated (n = 2) 4 × 3 incomplete Latin square design with 39-d periods. Treatments were: low (6.0%; LP), moderate (13.0%; MP), and high (20.0%; HP) crude-protein diets, containing 1.27 Mcal NE_L/kg dry matter. Diets were based on approximately 66% wheat straw and various proportions of ground corn grain and soybean meal. The estrous cycles of the heifers were synchronized, and 14 d after behavioral estrus, heifers received PGF_2α injections. After a further 40 h, at d 39 of each period, follicular fluid (FF) was aspirated from follicles of diameter >7 mm. The intake of the LP diet was 9% lower than that of MP and HP diets. Rumen ammonia and plasma urea nitrogen concentrations were highest in the HP and lowest in the LP, with intermediate levels in MP diets. No differences were found between treatments in plasma and FF concentrations of glucose and nonesterified fatty acids. High-protein diets increased urea concentrations very similarly in preovulatory FF and in plasma. No differences were observed between diets, in preovulatory follicle diameters and concentrations of androstenedione. However, higher estradiol and progesterone concentrations in FF were observed under the HP than under the MP diet, with no difference between diets in estrogen to progesterone ratio. It can be concluded that high concentrations of urea in plasma, caused by high dietary crude protein, penetrated into preovulatory follicles, but did not impair preovulatory characteristics. This lack of detrimental effects might be attributed to the use in this study of nonlactating heifers, which have fewer nutritional and physiological constraints and eliminate negative effects of potential interactions with high urea on dairy cows’ reproductive systems.     

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.     

Low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows

Objectives were to determine the effect of progesterone (P4) concentration on fertility of lactating dairy cows induced to ovulate follicles of the first follicular wave. Lactating dairy cows (n=989) at 38±3d postpartum were balanced by parity and body condition score and randomly assigned to 3 treatments: first follicular wave (FFW), first follicular wave with exogenous P4 (FFWP), or second follicular wave (SFW). All cows had their estrous cycle presynchronized with 2 injections of prostaglandin (PG) F(2α) given 14 d apart. Cows in the FFW and FFWP treatments started the ovulation synchronization protocol 3 d after the last PGF(2α) of the presynchronization protocol, whereas SFW cows received a GnRH injection (100 μg of gonadorelin diacetate; Cystorelin, Merial Ltd., Duluth, GA) 3 d after the last PGF(2α) of the presynchronization protocol and started the synchronization protocol 7 d later. The synchronization protocol consisted of GnRH on d -10, PGF(2α) on d -3, and GnRH concurrent with timed artificial insemination (AI) on d 0. Cows in the FFWP treatment received 2 controlled internal drug release inserts containing 1.38 g of P4 from d -8 to -3. Progesterone concentration was determined on d -10, -8, -6, -3, and 0 from all cows and at 7, 14, and 21 d after AI from a subsample of cows (n=170). Cows (n=715) had their ovaries scanned by ultrasound on d -10, -3, and 7 d. Pregnancy was diagnosed at 38 and 66 d after AI. Concentration of P4 from study d -8 to -3 was lowest for FFW cows (1.4±0.1 ng/mL) and similar between SFW (3.7±0.2 ng/mL) and FFWP (3.7±0.1 ng/mL) cows. Diameter of the dominant follicle on study d -3 was greater for FFW cows (16.5±0.3 mm) than for SFW cows (15.4±0.3 mm), but diameter of the dominant follicle of FFWP cows was not different (15.9±0.3 mm) compared with that of SFW and FFW cows. The incidence of multiple ovulation was largest for FFW cows (SFW=19.5, FFW=33.6, FFWP=19.0%), but pregnancy per AI (P/AI) at 66 d was smallest for FFW cows (SFW=38.9, FFW=22.3, FFWP=32.0%). Anovular cows in the SFW (19.4 vs. 42.8%) and FFWP (22.1 vs. 37.2%) treatments had reduced P/AI compared with cyclic cows, despite having similar or greater P4 concentration from study d -8 to -3, respectively. Estrus and ovulation synchronization protocols for lactating dairy cows must result in growth of ovulatory follicle under P4 concentration >2 ng/mL to ensure high P/AI.     

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous Holstein cows

Our objective was to evaluate the effect of manipulating progesterone (P4) concentrations before timed artificial insemination (TAI) on reproductive and endocrine outcomes in high-producing Holstein cows. Multiparous lactating Holstein cows (n = 80) were synchronized for first TAI using a Double-Ovsynch protocol and were randomly assigned to receive 25 mg of PGF_2α 1 d after the first GnRH treatment of the Breeding-Ovsynch protocol that included a once-used P4 insert (low-P4 group) or to receive 2 new P4 inserts during the Breeding-Ovsynch protocol (high-P4 group). Blood samples were collected thrice weekly from ?10 to 32 d relative to TAI for all cows and from 32 to 67 d after TAI for pregnant cows and were analyzed for P4 and pregnancy-specific protein B (PSPB) concentrations. Expression of IFNτ-stimulated gene 15 (ISG15) was assessed in blood leukocytes 18 and 20 d after TAI. As expected, P4 concentrations were greater for high-P4 cows than for low-P4 cows from 3 to 8 d before TAI. Incidence of double ovulation was 3-fold greater for low-P4 cows than for high-P4 cows (33 vs. 10%), which resulted in more twin pregnancies 32 d after TAI for low-P4 cows than for high-P4 cows (29 vs. 0%). Low-P4 cows had larger preovulatory follicles at the last GnRH treatment of the Double-Ovsynch protocol and greater P4 concentrations than high-P4 cows after TAI. Relative expression of ISG15 mRNA 18 and 20 d after TAI was greater for low-P4 cows than for high-P4 cows and for pregnant cows than for nonpregnant cows. Overall, PSPB concentrations tended to be greater for low-P4 cows than for high-P4 cows, and pregnant cows had greater P4 concentrations than nonpregnant cows. In summary, cows with low P4 before TAI had increased preovulatory follicle diameter, PSPB concentrations, relative expression of ISG15 mRNA 18 and 20 d after TAI, double ovulations, and twinning compared with cows with high P4 before TAI. Increasing P4 before TAI may effectively decrease double ovulation and twinning in high-producing multiparous Holstein cows.     

Ovarian follicular responses to high doses of pulsatile luteinizing hormone in lactating dairy cattle

Two experiments in lactating dairy cows examined ovarian follicular responses to high, frequent doses of exogenous LH pulses at levels associated with follicular cysts. In Experiment 1, estrus was synchronized in 12 cyclic lactating cows >40 d postpartum. Emergence of the second follicular wave (d 0) was determined by ultrasonography. Starting on d 1, cows received LH (40 microg/h; n = 7) or saline (2 mL/h; n = 5) in hourly pulses for up to 5 (n = 5) or 7 (n = 7) d. On d 2, all cows received two injections of PGF2alpha, 12 h apart. In experiment 2, 14 lactating cows (7 to 12 d postpartum) received LH (40 microg/h; n = 7) or saline (1 mL/h; n = 7) in hourly pulses for 7 d, beginning 24 h after start of the first follicular wave. Daily samples were used to determine serum concentrations of progesterone (P4), estradiol-17beta (E2), LH, and FSH. Profiles of LH were determined from blood samples collected at 12-min intervals for 8 h on d 3. During infusion of LH, serum P4 and FSH were similar across treatments in both experiments. Serum E2 concentrations were similar in experiment 1, but serum E2 was greater on d 2, 3, and 5 in LH-treated cows in experiment 2. Infusion increased LH pulse frequency and amplitude in both experiments. Formation of cysts did not differ between LH- and saline-treated cows in either experiment (1 of 7 vs. 0 of 5 and 1 of 6 vs. 0 of 7, respectively). Cows that ovulated had similar intervals to ovulation in experiment 1 [6.0 +/- 0.1 d (LH) vs. 6.4 +/- 0.2 d (saline)], but in experiment 2, ovulation was 14 d earlier in LH-treated cows (5.6 +/- 1.8 d vs 19.9 +/- 1.5 d). In conclusion, high concentrations of LH are not solely responsible for formation of cysts in lactating dairy cows. Pulsatile infusion of LH stimulated follicular growth and steroidogenesis and decreased time to first ovulation in anestrous postpartum cows.     

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.     

Effect of negative energy balance on the insulin-like growth factor system in pre-recruitment ovarian follicles of post partum dairy cows

Post partum negative energy balance (NEB) in dairy cattle is associated with a delayed return to ovarian cyclicity and reduced fertility. This study compared the IGF system of pre-recruitment ovarian follicles between cows in mild (n = 6) or severe (n = 6) NEB during early lactation. Ovaries were collected in the second week post partum, when circulating concentrations of IGF-I and glucose were lower (P < 0.01) in severe NEB cows. mRNA expression for IGF-II, type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1 to IGFBP-6 was determined by in situ hybridisation in individual follicles using radiolabelled oligonucleotide probes. Follicles were classified as very small (1-2.5 mm) or small (2.5-5 mm) and healthy or atretic. Relative mRNA concentrations were measured as optical density (OD) units using image analysis. Thecal IGF-II mRNA expression was highest in very small, healthy follicles (P < 0.05). Granulosa cell IGFBP-2 was the only component to change with EB status, with higher mRNA expression in mild compared with severe NEB cows (P < 0.05). IGFBP-1 and IGFBP-3 mRNA expression were undetectable. IGF-1R, IGFBP-4 and IGFBP-5 mRNA expression were not significantly altered by follicle size or health, but IGFBP-5 tended to increase in atretic follicles. The pattern of IGFBP-6 mRNA expression in theca paralleled that of IGF-II mRNA, with higher (P < 0.05) levels in healthy, very small follicles. In conclusion, the reduced expression of IGFBP-2 mRNA in severe NEB cows may alter the bioavailability of circulating IGF-I and locally produced IGF-II to modulate the pre-recruitment stages of follicles required to maintain normal post partum ovarian cyclicity.     

High feed intake increases liver blood flow and metabolism of progesterone and estradiol-17beta in dairy cattle

Increased liver blood flow (LBF) resulting from elevated feed intake in lactating dairy cows may increase steroid metabolism. Continuous infusion of bromosulphthalein (BSP; specifically metabolized in liver) was used to measure LBF. Similarly, progesterone (P4) and estradiol-17beta (E2) were administered by continuous infusion. Circulating concentrations at steady state were used to calculate the metabolic clearance rate (MCR) of BSP, P4, and E2. Experiment 1: Variation in LBF was determined in thee nonlactating and four lactating cows over 3 d at 3 to 5 h after feeding. Coefficients of variation ranged from 14 to 31% among cows within day and from 4 to 8% within cows across days. Experiment 2: Six nonlactating cows were used in a 3 x 3 Latin-square design with three feed regimens: no feed, 0.5 maintenance diet (M), and 1.5 M. Experiment 3: Eight lactating cows were used in a 4 x 4 Latin-square design with four feed regimens: no feed, 0.5 M, 1.5 M, and 2.2 M. In experiments 2 and 3, LBF and MCR of P4 increased immediately after feed consumption and increases persisted longer at higher intakes. The LBF reached a maximum at 2 h after feeding and MCR of P4 reached maximum at 3 h after feeding with a positive correlation (r = 0.92) between LBF and MCR for P4. Experiment 4: A crossover design was used to determine MCR of E2 in unfed or full-fed lactating dairy cows. The MCR of E2 increased immediately after feeding and stayed elevated throughout the 4.5-h infusion period. Thus, LBF and steroid metabolism were acutely elevated by feed consumption in lactating and nonlactating cows. Higher rates of LBF and steroid metabolism in lactating than in nonlactating cows may indicate chronic effects of higher feed intakes as well.     

Feeding rumen-inert fats differing in their degree of saturation decreases intake and increases plasma concentrations of gut peptides in lactating dairy cows

Our objective was to determine the effect of feeding rumen-inert fats differing in their degree of saturation on dry matter intake (DMI), milk production, and plasma concentrations of insulin, glucagon-like peptide 1 (7-36) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (CCK) in lactating dairy cows. Four midlactation, primiparous Holstein cows were used in a 4 × 4 Latin square experiment with 2-wk periods. Cows were fed a control mixed ration ad libitum, and treatments were the dietary addition (3.5% of ration dry matter) of 3 rumen-inert fats as sources of mostly saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), or polyunsaturated fatty acids (PUFA). Daily DMI, milk yield, and composition were measured on the last 4 d of each period. Jugular vein blood was collected every 30 min over a 7-h period on d 12 and 14 of each period for analysis of plasma concentrations of hormones, glucose, and nonesterified fatty acids. Feeding fat decreased DMI, and the decrease tended to be greater for MUFA and PUFA compared with SFA. Plasma concentration of GLP-1 increased when fat was fed and was greater for MUFA and PUFA. Feeding fat increased plasma glucose-dependent insulinotropic polypeptide and CCK concentrations and decreased plasma insulin concentration. Plasma CCK concentration was greater for MUFA and PUFA than for SFA and was greater for MUFA than PUFA. Decreases in DMI in cows fed fat were associated with increased plasma concentrations of GLP-1 and CCK and a decreased insulin concentration. The role of these peptides in regulating DMI in cattle fed fat requires further investigation.     

Prepartum supplementation of selenium and vitamin E to dairy cows: assessment of selenium status and reproductive performance

Incidence of retained placenta in dairy cows was evaluated in 627 parturitions. The herd was divided prepartum into three groups: 1) control, no treatment (n = 217 cows); 2) cows injected intramuscularly (n = 190) 21 to 10 d prior parturition with 45 mg Se and 2040 IU of vitamin E; and 3) cows intraruminally administered (n = 220) with two 30-g pellets containing 10% elemental selenium 2 mo prior to expected calving. Incidence of retained placenta (22.1%) was not reduced by Se in combination with vitamin E injection or intraruminal Se pellet nor were other measures of reproduction improved for cows fed a prepartum diet adequate in Se. At parturition the blood plasma Se concentrations were higher in treated postpartum with Se than in untreated cows. No difference in blood plasma Se was observed at parturition between cows with or without placenta retention. Cows dosed intraruminally with Se had a significant increase in milk Se, but this was too small to be a danger to human health. The present results on placenta retention suggest that this disorder is not a Se responsive disease in the dairy cow.     

Ovarian structures and circulating steroids in heifers and lactating cows in summer and lactating and dry cows in winter

Two experiments compared follicular and luteal development and circulating steroid concentrations from induced luteolysis to ovulation in lactating Holstein cows (n = 27; 40.0 +/- 1.5 kg milk/day) vs. nulliparous heifers (n = 28; 11 to 17 mo-old) during summer (Experiment 1), and in lactating (n = 27; 45.9 +/- 1.4 kg milk/d) vs. dry cows (n = 26) during winter (experiment 2). All females received PGF2,, 6 d after ovulation and were monitored until next ovulation by daily ultrasound and assay of serum progesterone (P4) and estradiol (E2). Every female was used two or three times. In Experiment 1, lactating cows had high incidence of multiple ovulation (63.5%) compared with heifers (1.3%). Among single ovulators, there was no difference in maximal size of ovulatory follicles between lactating cows and heifers (15.8 vs. 16.5 mm, respectively). However, lactating cows had lower peak serum E2 (8.6 vs. 12.1 pg/ml), took longer to ovulate after luteolysis (4.6 vs. 3.8 d), developed more luteal tissue volume (7,293.6 vs. 5,515.2 mm3), and had lower serum P4 on d 6 after ovulation (2.0 vs. 3.0 ng/ml) than heifers (data included multiple ovulators). In experiment 2, multiple ovulations were similar between lactating and dry cows (17.9 vs. 17.2%, respectively). Peak serum E2 was also similar between lactating and dry cows (7.6 vs. 8.5 pg/ml) although lactating cows had larger ovulatory follicles (18.6 vs. 16.2 +/- 0.4 mm). Lactating cows took longer to ovulate (4.8 vs. 4.2 d), developed more luteal tissue (7,599 vs. 5,139 +/- 468 mm3), but had similar serum P4 (2.2 vs. 1.9 ng/ ml) compared with dry cows. Therefore, lactating cows had similar or lower circulating steroid concentrations than dry cows or heifers, respectively, despite having larger ovarian structures.     

Effect of grain source and exogenous phytase on phosphorus digestibility in dairy cows

Two experiments were conducted to determine P digestibility in lactating dairy cows fed corn or barley as grain sources. The first experiment utilized a replicated incomplete 5 × 4 Latin square design with 8 lactating Holstein cows fed diets containing either corn alone or corn in combination with one of 4 barley varieties that differed in chemical composition. Total tract digestibility of P ranged from 11 to 29% for diets containing the barley varieties and was approximately 35% for the corn diet. A second experiment compared P digestibility in cows fed diets containing corn or barley when exogenous phytase was added to the diets. Lactating Holstein cows (n = 16) were arranged in 4 replications of a Latin square with 2 grains (barley or corn), fed separately or with added exogenous phytase (427 phytase units/kg of total mixed ration and 4 periods of 21 d. Phytate P comprised about 50% of the total P (0.46% P) in the total mixed ration. The concentration of serum inorganic P was higher in cows fed diets with exogenous phytase (5.8 vs. 6.5 mg/dL in cows fed barley diets and 5.5 vs 6.0 mg/dL in cows fed corn diets). Using acid detergent lignin as an internal marker, hydrolysis of phytate P was increased by the exogenous phytase, and total P digestibility tended to be increased. In contrast to Experiment 1, in Experiment 2 there was no effect of grain source on P digestibility and total fecal P. Dry matter intake and efficiency of milk production were not affected by exogenous phytase or grain type. Although phytase activity occurs in the rumen, physical properties of the diet and ruminal passage rates may prevent total hydrolysis of phytate in the rumen of lactating cows. Thus, exogenous dietary phytase might improve P digestibility in dairy cows in some dietary situations.