Effects of maternal obesity in an ovine model on metabolic outcomes in F2 adults and F3 neonates.

Accumulating evidence suggests that indications of metabolic syndrome can be inherited through the germline as a result of maternal obesity. We hypothesized that diet-induced maternal obesity during gestation would program metabolic consequences for multiple generations of offspring, even when first, second, and third generation offspring (F1, F2, F3, respectively) were fed only to requirements. Control (CON) and obese (OB) ewes (generation 0; F0) were bred to a single ram to produce the first generation of offspring (F1). From 60 d prior to conception through term, CONF0 ate 100% National Research Council recommendations (NRC), while OBF0 ewes ate 150% NRC. All F1, F2, and F3 ate 100% NRC after weaning. All mature F1 ewes were bred to a single ram to generate CONF2 (n = 6) and OBF2 (n = 10). All mature F2 ewes were bred to a single ram to produce CONF3 (n = 6) and OBF3 (n = 10). OBF2 ewes exhibited greater (P < 0.0001) plasma cortisol than CONF2 throughout gestation. A glucose tolerance test at 90% gestation revealed OBF2 ewes had higher (P < 0.05) insulin response with similar glucose, resulting in greater (P < 0.05) insulin resistance. OBF3 neonates had similar weight, lean mass, and body fat mass to CONF3 neonates. These data suggest that multigenerational programming of adverse metabolic phenotypes occur in association with F0 maternal obesity, yet adiposity may return to CON levels in F3 neonates.

Intergenerational impact of maternal overnutrition and obesity throughout pregnancy in sheep on metabolic syndrome in grandsons and granddaughters.

We previously reported that maternal overnutrition and obesity (MO) throughout pregnancy and lactation in sheep (MOF0) decreases term fetal pancreatic ß-cell numbers and increases perirenal adiposity producing hyperphagia, increased adiposity and insulin resistance in adult female offspring (MOF1) fed ad libitum. Pregnant female MOF1 exhibited increased blood glucose from mid to late gestation vs control F1 (CTRF1) though both groups ate only to NRC recommendations. MOF1 ewes delivered female offspring (F2) who like their MOF1 mothers exhibited increased abdominal adiposity and absent neonatal leptin surge. In the current work, we determined if adult MOF2 exhibited metabolic syndrome components when fed ad libitum. After weaning, MOF2 males (n = 5), MOF2 females (n = 6), CTRF2 males (n = 5), and CTRF2 females (n = 6) were fed to NRC requirements until 19 mo followed by 12-wk ad libitum feeding. Body weight and % fat increased (P < 0.01) in all F2 during this feeding trial. MOF2 males were heavier (P < 0.01) than CTRF2 males and females, and MOF2 females throughout the trial. By wk 8, baseline blood glucose concentrations increased (P < 0.001) in MOF2 females, but not other groups, remaining elevated throughout the trial. Baseline insulin was similar through wk 6, increasing (P < 0.05) at wk 8 in MOF2 females only. MOF2 female insulin returned to CTRF2 female levels during wk 10 and 12. The progressive increase of plasma glucose on wk 8 in association with increased insulin in MOF2 females but not other groups demonstrated a diet-induced increase (P < 0.001) in MOF2 female insulin resistance. The subsequent decline in insulin during wk 10 and 12 despite elevated glucose in MOF2 females is consistent with a decrease in glucose-stimulated pancreatic ß-cell function. These data indicate that ad libitum feeding exceeds the pancreatic secretory response predisposing MOF2 females to hyperglycemia. Furthermore, there was a sex difference where MOF2 males increased body mass and MOF2 females displayed insulin/glucose dysregulation.

Intravaginal probiotics modulated metabolic status and improved milk production and composition of transition dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of probiotics (a lactic acid bacteria cocktail) around parturition would influence metabolic status and increase milk production of transition dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of the 3 experimental groups receiving intravaginal infusion of probiotics or carrier (i.e., sterile skim milk) once a week at wk -2, -1, and +1 relative to calving as follows: 2 consecutive probiotics before parturition and 1 carrier dose after parturition (TRT1), 3 consecutive probiotics doses around parturition (TRT2), and 3 consecutive carrier doses around parturition (CTR). The probiotics were a lyophilized culture mixture composed of FUA3089 and FUA3138 and FUA3140 with a cell count of 10 to 10 cfu/dose. Blood was sampled from wk -2 to +3 and milk was sampled on the third day in milk (DIM) and from wk +1 to +5 on a weekly basis. Feed intake and milk production was monitored until wk +8. Results showed that the TRT2 group (366.12 ± 49.77 µmol/L) had a lower ( = 0.01) concentration of NEFA in the serum than the CTR group (550.85 ± 47.16 µmol/L). The concentrations of IgG in the milk were 32.71 ± 3.00 mg/mL in the TRT1 group, 17.47 ± 4.54 mg/mL in the TRT2 group, and 6.73 ± 3.43 mg/mL in the CTR group at 3 DIM ( < 0.01). Meanwhile, both the TRT1 and the TRT2 group had lower haptoglobin in the milk compared with the CTR group at 3 DIM ( < 0.01). The TRT1 group had greater milk protein content than the CTR group (2.99 ± 0.04 vs. 2.82 ± 0.04%; = 0.02), whereas the TRT2 group tended to have greater lactose content compared with the CTR group (4.53 ± 0.03 vs. 4.44 ± 0.03%; = 0.05). The effect of treatment interacted with parity with regards to milk production and feed efficiency. Multiparous cows in the TRT1 and TRT2 groups had greater milk production and feed efficiency than those in the CTR group ( < 0.01 and = 0.02, respectively). Among primiparous cows, those in the TRT2 group had greater milk production ( = 0.04) whereas those in the TRT1 group had lower feed intake ( < 0.01) than those in the CTR group. Both the TRT1 and the TRT2 groups had enhanced feed efficiency compared with the CTR group ( < 0.01). In conclusion, intravaginal infusion of lactic acid bacteria modulated concentrations of selected serum metabolites and milk components and increased milk efficiency of transition dairy cows.

Intravaginally administered lactic acid bacteria expedited uterine involution and modulated hormonal profiles of transition dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of lactic acid bacteria (LAB) around parturition could expedite involution rate of the uterus and improve reproductive performance of postpartum dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of 3 experimental groups: (1) 1 dose of LAB in wk -2 and -1 and 1 dose of carrier in wk 1 relative to the expected day of parturition (TRT1); (2) 1 dose of LAB in wk -2, -1, and 1 (TRT2); and (3) 1 dose of carrier in wk -2, -1, and 1 (CTR). The LAB treatment was a lyophilized mixture of Lactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 with a cell count of 10(8) to 10(9) cfu/dose. Uterine involution and ovarian activity was evaluated by transrectal ultrasonography weekly from d 7 to 49 postpartum. Blood samples were collected from a subset of cows to quantify prostaglandin (PG) F2α metabolite (PGFM), PGE2, and progesterone. Cows treated with LAB had smaller cross-sectional areas of gravid horn and uterine body on d 14 postpartum. Cows in TRT2 resumed ovarian cyclicity earlier, as indicated by increased concentrations of serum progesterone. Cows in TRT1 had fewer days open than those in the CTR (110 vs. 150 d), whereas cows in TRT2 and CTR did not differ in days open. In addition, both TRT1 and TRT2 increased the concentrations of PGFM at calving week, and cows in TRT2 also had greater concentrations of PGE2 on d 14 and d 21 postpartum relative to CTR. Overall, cows treated intravaginally with LAB had smaller gravid horn and uterine body on d 14 postpartum than those in the CTR group. Treatment with LAB also increased concentrations of serum PGFM (3,533±328pg/mL in TRT1, 4,470±372pg/mL in TRT2, and 2,000±328pg/mL in CTR on d 0, respectively), with the TRT1 group having fewer cows that resumed ovarian cyclicity but fewer days open compared with both TRT2 and CTR groups. More research is warranted to better understand the mechanism(s) by which intravaginal LAB expedited uterine involution and affected hormonal profiles.

Multigenerational impact of maternal overnutrition/obesity in the sheep on the neonatal leptin surge in granddaughters.

BACKGROUND/OBJECTIVES: We have reported that maternal overnutrition/obesity (OB) in sheep resulting from feeding 150% of National Research Council (NRC) requirements throughout gestation leads to maternal hyperglycemia and hyperinsulinemia. Further, newborn lambs born to OB vs control-fed (CON, 100% of NRC) ewes exhibited greater adiposity, increased blood cortisol, insulin and glucose and the elimination of the postnatal leptin spike seen in lambs born to CON ewes. This early postnatal leptin peak is necessary for the development of hypothalamic circuits, which program appetite in later life. This study evaluated the multigenerational impact of OB on insulin:glucose dynamics of mature female F1 offspring fed only to requirements throughout gestation and on their lambs (F2 generation). DESIGN AND METHODS: Adult F1 female offspring born to OB (n=10) or CON (n=7) ewes were utilized. All F1 ewes were subjected to a glucose tolerance test at midgestation and late gestation. Jugular blood was obtained from F2 lambs at birth (day 1) through postnatal day 11, and plasma glucose, insulin, cortisol and leptin concentrations were determined. Dual-energy X-ray absorptiometry was utilized to determine bone mineral density, bone mineral content, lean tissue mass and fat tissue mass. RESULTS: Fasted blood glucose and insulin concentrations were greater (P<0.05) in OBF1 than CONF1 ewes at midgestation and late gestation. Further, after glucose infusion, both glucose and insulin concentrations remained higher in OBF1 ewes (P<0.05) than CONF1 ewes, demonstrating greater insulin resistance. Blood concentrations of glucose, insulin and cortisol and adiposity were higher (P<0.01) in OBF2 lambs than CONF2 lambs at birth. Importantly, OBF2 lambs failed to exhibit the early postnatal leptin peak exhibited by CONF2 lambs. CONCLUSIONS: These data suggest that these OBF2 lambs are predisposed to exhibit the same metabolic alterations as their mothers, suggesting a multigenerational programming effect.

Intravaginal administration of lactic acid bacteria modulated the incidence of purulent vaginal discharges, plasma haptoglobin concentrations, and milk production in dairy cows.

This investigation studied the effects of intravaginal administration of a mixture of lactic acid bacteria (LAB) on the incidence of purulent vaginal discharges (PVD), plasma haptoglobin concentrations, and milk production in dairy cows. A total of 82 pregnant primiparous and multiparous Holstein dairy cows were used in this study. Half of the cows received intravaginally 1mL of LAB at 10(10)-10(12)cfu/mL and the other half 1mL of reconstituted skim milk (i.e., carrier) (controls). Administration of LAB was conducted once per wk during 2 and 1wk before the expected day of calving and at 1, 2, 3, and 4wk postpartum. Data demonstrated that intravaginal administration of LAB decreased the occurrence of PVD at 3wk postpartum (P<0.05). Concentrations of plasma haptoglobin, an acute phase protein often associated with uterine infections, was lower in cows treated with the LAB mixture at 2wk (P<0.001) and 3wk (P<0.05) postpartum. Treatment with LAB did not improve overall pregnancy rate, but the treated multiparous cows produced more milk than their control counterparts (P<0.05), whereas no difference was observed in primiparous cows regarding milk yield (P>0.05). Overall, this is the first study demonstrating that intravaginal LAB administration lowers the incidence of PVD and enhances milk production in dairy cows. Further research is warranted to evaluate the effects of LAB on reproductive performance in a larger cohort of cows.

Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.

Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1.

Reduced capability of the uterus to support pregnancy in the absence of its interaction with secretions from male accessory glands has been demonstrated in rodents and to some extent in pigs. However, in cattle, the role of postmating inflammatory response on pregnancy success has not been studied. The current study examined the influence of uterine presensitization with seminal antigens at breeding on pregnancy outcome in cows. Lactating beef (n=1090) and dairy (n=800) cows received 0.5 mL seminal plasma (SP), 40 ng recombinant human transforming growth factor-beta1 (rhTGF-beta1), or 0.5 mL bovine serum albumin (BSA), or were left untreated before or at insemination. Semen was deposited into the anterior cervix using a second insemination gun. Pregnancy was diagnosed at 35 to 40 d postinsemination by transrectal ultrasonography or from records of calves born the subsequent calving season. Pregnancy rates in beef cows did not differ among treatments but differed among trials (69.8%, 52.5% vs. 40.3%; P<0.05). In trials where average pregnancy rates were below 50%, treatments with TGF-beta1 but not SP tended (P<0.07) to increase pregnancy rates in beef cows. In dairy cows, SP and TGF-beta1 improved pregnancy outcome by 10 percentage points, but these increments did not achieve statistical significance. In conclusion, this study did not find any conclusive evidence for the effect of TGF-beta1 or seminal plasma on pregnancy outcome in lactating dairy or beef cows but realized marginal improvements when pregnancy rates were below 50% (compromised fertility).

Effect of weaning regimen on energy profiles and reproductive performance of beef cows.

The effect of shifting calf-weaning age on profiles of energy status (BW, BCS, and rib and rump fat) and reproductive performance of beef cows was evaluated in a 3-yr study. Pregnant and lactating crossbred beef cows (n = 408), mainly of Angus and Hereford breeding, were stratified by age and by sex and BW of their calves and assigned randomly into 2 treatments: weaning at approximately 180 d (early weaning) and normal weaning 45 d later (control). Cows were managed together on native range pastures and supplemented with harvested forage during the winter months. Cow BW, BCS, rib fat, and rump fat were measured periodically from early weaning through the next breeding. Reproductive performance was evaluated by calving intervals (CI), days from initiation of breeding to calving (BCI), retention in the herd, and adjusted 205-d weaning BW of the subsequent calf. Early weaned cows had greater (P < 0.001) BW at normal weaning than control cows, but the overall pattern of cow BW did not differ (P > 0.05) among treatments. Peak and nadir BCS occurred at precalving and postcalving periods, respectively and were greater (P < 0.001) at each period in early weaned than in control cows and in cows > or =5-yr-old than in younger cows. Patterns for rib fat and rump fat were nearly identical to those of BCS except for the 3-way interaction (P < 0.001) of treatment, age, and period on rump fat. Mean CI (372.4 +/- 2.1 d) and BCI (299.7 +/- 1.9 d) were not affected (P = 0.42) by treatment but varied (P < 0.001) with age of the cow. Age of cow accounted for 16% of total variation in CI and 12% of total variation in gestation length (P < 0.001). The intervals were longer (P < 0.001) in primiparous cows than in older cows. Early weaning decreased risk of culling in cows and thereby increased (P < 0.05) overall persistence by 11% over control cows. Earlier weaning of cows in the previous year increased (P < 0.001) weaning weight of the subsequent calf by 8.6 kg per cow per yr. Shifting weaning time increased storage of consumed energy as evidenced by increased rump fat, for use later during high-energy demand, ultimately improving overall productivity of the cow-calf system.