Association between leptin single nucleotide polymorphism and reproductive performance of lactating Holstein cows

Animal Reproduction Science 127 (2011) 126–134

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Association between leptin single nucleotide polymorphism and reproductive performance of lactating Holstein cows R.C. Chebel a , J.E.P. Santos b,∗ a b

Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN 55108, USA Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA

a r t i c l e

i n f o

Article history: Received 21 February 2011 Received in revised form 7 June 2011 Accepted 14 June 2011 Available online 16 August 2011 Keywords: Dairy cow Leptin genotype Reproduction SNP

a b s t r a c t The objectives of the current study were to evaluate the associations among reproductive performance of Holstein cows, single nucleotide polymorphism (SNP) in the R4C locus of the Exon-2 region of the leptin gene and health disorders. Lactating dairy cows (n = 814) had their DNA sequenced at the Exon-2 region of the leptin gene to determine the presence of SNP in the R4C locus. Cows had the stage of estrous cycle synchronized with two injections of PGF2␣ 14 d apart, with or without a progesterone insert. After the second PGF2␣ , cows were either inseminated on detected estrus and, if not inseminated within 13 d, cows were submitted to a timed artificial insemination (AI) protocol 13 d after the second PGF2␣ . Blood was sampled at 35 ± 3, 49 ± 3, and 62 ± 3 d in milk (DIM) and cows with progesterone <1.0 ng/mL in the first two samples were considered to be anovular, whereas those with at least one of the first two samples with progesterone ≥1.0 ng/mL were considered to have initiated estrous cyclicity. Anovular cows with progesterone ≥1.0 ng/mL at 62 ± 3 DIM were considered to have resumed estrous cyclicity. Pregnancy was diagnosed at 31 and 60 d after first postpartum AI and at 42 d after subsequent inseminations. Resulting genotypes were CC (34.6%), CT (48.2%), and TT (17.2%). Leptin genotype was associated (P = 0.03) with increased prevalence of estrous cyclic cows at 49 ± 3 DIM, as TT cows were and tended to be less likely to be estrous cyclic than CC and CT cows, respectively. Other measures of reproductive performance in the first 305 DIM were not associated with leptin genotype. Cows diagnosed with at least one postparturient disease were less likely to become pregnant after first (P < 0.01) and second (P = 0.02) AI and to be pregnant at 305 DIM (P < 0.01). Furthermore, cows diagnosed with a disease event had a 36% reduction (P < 0.001) in the rate of pregnancy, which extended the median interval to pregnancy by 60 d. Estrous cyclic cows had a 35% increased rate of pregnancy, which reduced the interval to pregnancy by 34 d. Cows homozygous for the T allele were less likely to be cyclic early postpartum, but leptin genotype was not associated with other reproductive responses when cows were subjected to a controlled breeding program. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Energy status of dairy cattle during the postpartum period is associated with reproductive performance

∗ Corresponding author. Tel.: +1 352 392 1958. E-mail address: jepsantos@ufl.edu (J.E.P. Santos). 0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2011.06.011

(Santos et al., 2009). Resumption of normal ovulatory cycles is associated with the timing of nadir of energy balance in early lactation (Butler, 2000). Both, body condition score (BCS) and BCS changes at first AI postpartum were associated with prevalence of estrous cyclic cows by 65 DIM and fertility to the first postpartum AI (Santos et al., 2009). Leptin, a peptide hormone secreted mainly by white adipose tissue (Zhang et al., 1994), regulates feed intake,

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energy expenditure, and reproductive and immune functions (Nkrumah et al., 2005; Cunningham et al., 1999; Lord et al., 1998). Reduced leptin concentrations are observed in animals with depleted fat reserves, indicating that a state of negative energy balance is occurring (Chilliard et al., 2005). In such cases, the prioritized use of available calories is for functions that assure short-term survival and not reproductive functions, which are of lesser importance to the animal. The substitution of a cytosine (C) by a thymidine (T) nucleobase in the R4C locus of the Exon-2 region of the leptin gene, characteristic of a SNP, which has been suggested to alter the tertiary conformation of the leptin protein by adding an unpaired cysteine to its structure, altering the affinity of the hormone to its receptors (Konfortov et al., 1999; Liefers et al., 2003, 2005). Polymorphism of the R4C locus of the leptin gene has been associated with changes in leptin concentrations prepartum in dairy cows, as cows homozygous for the T allele have smaller concentrations than those homozygous for the C allele (Liefers et al., 2003). Energy status, degree of fatness, and tissue responsiveness to leptin all have been implicated with changes in leptin concentrations in mammals, including cattle. Because cattle homozygous for the T allele in exon 2 of the leptin gene had greater body fatness compared with those homozygous for the C allele (Buchanan et al., 2002), it is unlikely that degree of fatness induced changes in leptin. Therefore, it is possible that the SNP in the R4C locus of the Exon-2 region of the leptin gene could alter the activity of the leptin hormone. This, consequently, could influence reproductive performance of lactating dairy cows. Although SNP of the coding region for the leptin gene have been associated with lactation and health of dairy cows (Liefers et al., 2002; Chebel et al., 2008), little is known about the reproductive performance of dairy cows bearing the different alleles. Liefers et al. (2002) found no association between SNP of the leptin gene and interval to first postpartum ovulation. Komisarek and Antkowiak (2007) screened 219 Jersey cows and observed that those bearing the TT genotype in the A59V locus had a shorter calving interval than cows bearing the CC and CT alleles. These results suggest that selection of cattle to increase the prevalence of specific SNP of the leptin gene might benefit reproduction of dairy cows. The hypothesis of the present study was that SNP at the R4C locus of the Exon-2 region of the leptin gene is associated with resumption of postpartum ovulation and fertility of lactating dairy cows and these associations are independent of incidence of health disorders. Therefore, the objectives of the present study were to characterize the reproductive performance of cows with SNP in the R4C locus in the Exon-2 region of the leptin gene and cows that experienced disease during their lactation.

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in the dairy during the study period and the rolling herd milk production average was 12,035 kg of 3.5% fatcorrected milk. Facilities, management, and diets have been described elsewhere (Chebel et al., 2008). 2.2. Reproductive management All cows received two injections of 25 mg of PGF2␣ (dinoprost tromethamine; 5 mL Lutalyse Sterile Solution; Pfizer Animal Health, Madison, NJ), at 35 ± 3 and 49 ± 3 DIM, and 521 of the 814 cows received a controlled internal drug-release (CIDR) insert containing 1.38 g of progesterone (EAZI-BREED CIDR cattle insert; Pfizer Animal Health, Madison, NJ) from 42 ± 3 to 49 ± 3 DIM. Starting at 49 ± 3 DIM, 555 cows were observed for estrus and inseminated and those not inseminated by 62 ± 3 DIM were submitted to a timed AI protocol, whereas the remaining 259 cows were not inseminated on estrus and were all submitted to the timed AI protocol at 62 ± 3 DIM. The timed AI protocol consisted of an injection of 100 ␮g GnRH (gonadorelin diacetate tetrahydrate; 2 mL Cystorelin; Merial, Ltd, Iselin, NJ), 7 d later an injection of PGF2␣ , 2 d later a second injection of GnRH, and 24 h later timed AI. After the first postpartum AI, 294 cows received a CIDR insert from 14 ± 1 to 21 ± 1 d after AI and 520 cows received no further treatment. 2.3. Estrous detection and artificial insemination Cows were observed daily, in the morning, for estrus based on removal of tail paint (All-weather Paintstick, LA-CO Industries, Chicago, IL) and those in estrus were inseminated on the same morning. One technician inseminated cows 6 d a week, which coincided with the majority of AI, and a relief technician inseminated cows once a week. 2.4. Body condition scoring Cows were scored for body condition using a 1 to 5 scale at 3 ± 3, 35 ± 3, and 62 ± 3 DIM as described by Ferguson et al. (1994). For purpose of statistical analysis cows with BCS ≤ 2.75 were classified as having lesser BCS, cows with BCS between 3.00 and 3.75 were classified as having moderate BCS, and those with BCS ≥ 4.00 were classified as having greater BCS. Because there were only a few cows with BCS ≥ 4.0 at 62 ± 3 DIM, they were grouped with cows that had BCS between 3.0 and 3.75 for statistical analysis. Cows were also classified according to the amount of BCS lost from 3 ± 3 to 62 ± 3 DIM as excessive when BCS loss was ≥ 1.0 unit, moderate when BCS loss was between 0.25 and 0.75 units, and no loss when no change in BCS was observed or when cows had an increase in BCS. 2.5. Incidence of health disorders

2. Materials and methods 2.1. Animals, housing, diets, and management Lactating Holstein cows, 458 multiparous and 356 primiparous, from a commercial dairy farm were used in this study. Approximately 3200 lactating cows were housed

Health disorders that occurred during the lactation such as retained fetal membranes, displacement of the abomasum, lameness, and mastitis were diagnosed and recorded as described by Chebel et al. (2008). For the current study, cows were classified as experiencing or not experiencing at least one of the diseases described above.

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2.6. Blood sample collection, leptin genotyping, progesterone analysis and classification of anovular condition

the number of cows pregnant at 305 DIM by the number of cows initially enrolled in the study. 2.8. Statistical analyses

Blood samples (7 mL) were collected from the median coccygeal vein or artery using evacuated tubes (Becton Dickinson, Franklin Lakes, NJ) containing K2 EDTA. Immediately upon collection, samples were placed in ice and transported to the laboratory within 5 h. For genotyping of the R4C locus of the Exon-2 region of the leptin gene, whole blood samples were sent to IGENITY (Lincoln, NE) and assayed as described in Chebel et al. (2008). Plasma from samples collected at 35 ± 3, 49 ± 3, and 62 ± 3 DIM were separated and frozen at -25 ◦ C and later analyzed for concentrations of progesterone using an ELISA procedure. The sensitivity of the assay was 0.05 ng/mL and the intra- and inter- assay CV were 5.7% and 8.1%, respectively. Cows with progesterone <1.0 ng/mL at 35 ± 3 and 49 ± 3 DIM were classified as anovular, and cows with at least one sample in which progesterone ≥1 ng/mL were classified as estrous cyclic. Cows classified as anovular by 49 ± 3 DIM that had progesterone ≥1 ng/mL at 62 ± 3 DIM were classified as experiencing resumption of estrous cyclicity.

2.7. Pregnancy diagnoses and calculation of reproductive outcomes Pregnancy was diagnosed at 31 ± 3 d after the first postpartum AI by ultrasonographic examination of the uterus and its contents, and it was characterized by visualization of an embryo. Cows diagnosed pregnant by ultrasonography were re-examined by palpation per rectum of the uterine contents 60 ± 3 d after AI. For cows inseminated more than once after parturition pregnancy was diagnosed by per rectum palpation of the uterus at 42 ± 3 d after AI, and cows re-inseminated before being examined for pregnancy were considered to be non-pregnant. Cows diagnosed not pregnant that had not been re-inseminated were treated to resynchronize the stage of the estrous cycle with the same timed AI protocol described previously. Proportion of cows inseminated on estrus between 49 ± 3 and 62 ± 3 DIM was calculated by dividing the number of cows receiving AI on detected estrus by the number of cows eligible to be inseminated on estrus. Because a group of cows was not eligible to be inseminated on estrus from 49 ± 3 and 62 ± 3 DIM, only 555 cows were used for calculation of AI submission rate. Pregnancy per AI (P/AI) was calculated by dividing the number of cows diagnosed pregnant by the number of cows receiving AI. Proportion of cows that experienced pregnancy loss from 31 ± 3 to 60 ± 3 d after the first postpartum AI was calculated by dividing the number of cows not pregnant at 60 ± 3 d by the number of cows diagnosed pregnant at 31 ± 3 d. Proportion of cows pregnant after first and second postpartum AI was calculated by dividing the total number of cows pregnant after first and second AI by the overall number of cows enrolled in the study. Finally, the proportion of cows that became pregnant by 305 DIM was calculated by dividing

Binary data were analyzed by logistic regression using the LOGISTIC procedure of SAS (SAS/STAT, SAS Institute Inc., Cary, NC, USA). The statistical model used for analysis of the proportion of cyclic cows at 49 ± 3 and 62 ± 3 DIM and the proportion of anovular cows that resumed estrous cyclicity by 62 ± 3 DIM included leptin genotype (CC, CT, and TT), parity (primiparous and multiparous), interaction between leptin genotype and parity. Models used for analysis of proportion of cows inseminated on estrus, P/AI, pregnancy loss, and proportion of cows pregnant at 305 DIM included leptin genotype (CC, CT, and TT), parity (primiparous and multiparous), estrous cyclic status at 49 DIM (anovular and cyclic), interaction between leptin genotype and parity, synchronization method, BCS at 3 ± 3, 35 ± 3, and 62 ± 3 DIM, BCS changes from 3 ± 3 to 62 ± 3 DIM, and occurrence of disease. The logistic regression models removed variables by a backward elimination based on the Wald’s statistics criterion if P > 0.15. Adjusted odds ratio (AOR) and 95% confidence intervals (CI) were calculated. The rate of pregnancy in the first 305 DIM was analyzed by the Cox proportional hazards ratio model using the PHREG procedure of SAS. Adjusted hazard ratio (AHR) and 95% CI were calculated. The hazard in this analysis determines the daily rate of pregnancy for cows in the first 305 DIM. The statistical model included leptin genotype (CC, CT, and TT), parity (primiparous and multiparous), the interaction between leptin genotype and parity, synchronization method, cyclic status, BCS at 3 ± 3, 35 ± 3, and 62 ± 3 DIM, BCS change from 3 ± 3 to 62 ± 3 DIM, and occurrence of disease. A backward elimination procedure was used and variables were retained in the model when P ≤ 0.15 according to the Wald statistics criterion. Variables identified as affecting pregnancy rate were further analyzed for their effect on the median and mean intervals between calving and pregnancy by survival analysis using the product limit method of the Kaplan-Meier model of the LIFETEST procedure of SAS. 3. Results As described by Chebel et al. (2008) the proportion of cows bearing the C and T allele were 82.8 and 65.4%, respectively, and the proportions of cows bearing CC, CT, and TT genotypes were 34.6, 48.2, and 17.2%, respectively. There was no difference (P = 0.98) in the distribution of cows with different leptin genotypes submitted to the different estrous synchronization protocols. The observed frequency of alleles in the study population fits the Hardy-Weinberg equilibrium based on the expected frequency. 3.1. Estrous cyclic status The proportion of cows classified as estrous cyclic at 49 ± 3 DIM was associated (P = 0.03) with the leptin genotype (Table 1), and TT cows were (P < 0.01) and tended

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Table 1 Reproductive performance of lactating Holstein cows according to leptin genotype. Item

Estrous cyclic at 49 ± 3 DIM,b % (n) Resumed cyclicity,c % (n) Cyclic at 62 ± 3 DIM,d % (n) DIM at 1st AI, mean ± SEM AI submission rate,e % (n) Pregnant 1st AI, % (n) 31 ± 3 d after AI 60 ± 3 d after AI Pregnancy loss 1st AI, % (n) Pregnant 2nd AI, % (n) Pregnant 1st and 2nd AI, % (n) Pregnant 305 DIM, % (n)

Leptin genotypea

P

CC

CT

TT

65.1a (275) 39.6 (96) 78.9 (275) 70.0 ± 1.7 49.5 (186)

59.8A (378) 38.8 (152) 75.4 (378) 69.7 ± 1.7 46.9 (271)

51.9b,B (135) 41.5 (65) 71.9 (135) 69.8 ± 1.8 43.9 (98)

0.03 0.90 0.25 0.87 0.70

33.7 (282) 26.2 (282) 22.1 (95) 24.5 (212) 44.3 (282) 79.4 (282)

33.4 (392) 27.3 (392) 18.3 (131) 32.3 (294) 51.3 (392) 76.3 (392)

31.4 (140) 23.6 (140) 25.0 (44) 23.9 (113) 42.9 (140) 75.7 (140)

0.99 0.80 0.48 0.25 0.18 0.54

a,b

Within rows, proportions with different superscript letters differ (P < 0.05). Within rows, proportions with different superscript letters tend to differ (P < 0.10). a Single nucleotide polymorphism in the R4C locus in the Exon-2 region of the leptin gene. b Estrous cyclic if progesterone ≥1.0 ng/mL in at least one of two blood samples collected at 35 ± 3 and 49 ± 3 DIM. c Anovular cows at 49 ± 3 DIM that had progesterone ≥1.0 ng/mL at 62 ± 3 DIM. d Estrous cyclic if progesterone ≥1.0 ng/mL in at least one of the blood samples collected at 35 ± 3, 49 ± 3, and 62 ± 3 DIM. e Proportion of cows inseminated on estrus from 49 ± 3 to 62 ± 3 DIM.

A,B

to be (P = 0.10) less likely to be estrous cyclic than CC and CT cows, respectively. A smaller (P < 0.01) proportion of primiparous cows was cyclic at 49 ± 3 DIM compared with multiparous cows (46.9 compared with 70.6%). Cows with moderate and greater BCS at 62 ± 3 DIM were more likely (P < 0.01) to be estrous cyclic at 49 ± 3 DIM compared with those with lesser BCS (66.9 compared with 55.5%). Cows diagnosed with at least one disease event postpartum were less likely (P < 0.001) to be estrous cyclic at 49 ± 3 DIM compared with those that did not experience disease (Table 2). The proportion of cows diagnosed as anovular at 49 ± 3 DIM that resumed estrous cyclicity by 62 ± 3 DIM was not associated with leptin genotype (Table 1). A greater (P = 0.05) proportion of anovular multiparous cows resumed estrous cyclicity by 62 ± 3 DIM compared with primiparous cows (43.5 compared with 36.8%). Body condition score at 62 ± 3 DIM was (P < 0.01) associated with the

proportion of cows resuming ovulation (lesser = 32.0, moderate/greater = 53.6%). Proportion of anovular cows that resumed estrous cyclicity by 62 ± 3 DIM also was influenced (P = 0.03) by occurrence of disease (Table 2). At 62 ± 3 DIM, leptin genotype was not associated with estrous cyclic status (Table 1). A greater (P < 0.01) proportion of multiparous cows was cyclic by 62 ± 3 DIM compared with primiparous cows (83.4 compared with 66.5%). Body condition score at 62 ± 3 DIM (lesser = 69.7, moderate/greater = 84.6%; P < 0.01) and occurrence of disease (P < 0.001; Table 2) influenced the prevalence of estrous cyclic cows at 62 ± 3 DIM. 3.2. Artificial insemination submission rate Average DIM at first postpartum AI and the proportion of cows inseminated on detected estrus between 49 ± 3 and 62 ± 3 DIM were not associated with lep-

Table 2 Reproductive performance of lactating Holstein cows diagnosed with at least one disease event postpartum. Item

Estrous cyclic at 49 ± 3 DIM,c % (n) Resumed estrous cyclicity,d % (n) Estrous cyclic at 62 ± 3 DIM,e % (n) DIM at 1st AI, LSM ± SEM AI submission rate,f % (n) Pregnant 1st AI, % (n) 31 ± 3 d after AI 60 ± 3 d after AI Pregnancy loss 1st AI, % (n) Pregnant 2nd AI, % (n) Pregnant 1st and 2nd AI, % (n) Pregnant at 305 DIM, % (n) a b c d e f

Diseasea

AOR (95% CI)b

P

No

Yes

62.2 (606) 42.8 (229) 78.4 (606) 67.6 ± 0.4 50.2 (424)

53.9 (182) 31.0 (84) 68.1 (182) 67.8 ± 0.7 37.4 (131)

1.84 (1.28–2.65) 1.91 (1.07–3.43) 2.30 (1.53–3.45) – 1.58 (1.03–2.46)

0.001 0.03 0.001 0.81 0.04

35.8 (628) 29.6 (628) 17.3 (225) 30.8 (455) 51.8 (628) 80.6 (628)

24.2 (186) 15.1 (186) 37.8 (45) 20.7 (164) 32.8 (186) 66.1 (186)

1.64 (1.12–2.40) 2.25 (1.45–3.51) 0.41 (0.20–0.83) 1.73 (1.11–2.69) 2.13 (1.49–3.04) 2.11 (1.45–3.08)

0.01 0.001 0.01 0.02 0.001 0.001

Retained fetal membranes, mastitis, lameness, or displacement of the abomasum. AOR = adjusted odds ratio; CI = confidence interval; cows diagnosed with at least one disease event were considered referent for comparison. Estrous cyclic if progesterone ≥ 1.0 ng/mL in at least one of two blood samples collected at 35 ± 3 and 49 ± 3 DIM. Anovular cows at 49 ± 3 DIM that had progesterone ≥ 1.0 ng/mL at 62 ± 3 DIM. Estrous cyclic if progesterone ≥1.0 ng/mL in at least one of the blood samples collected at 35 ± 3, 49 ± 3, and 62 ± 3 DIM. Proportion of cows inseminated on estrus from 49 ± 3 to 62 ± 3 DIM.

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tin genotype (Table 1). Multiparous cows had a shorter (P < 0.01) interval from calving to first AI compared with primiparous cows (71.2 ± 1.7 and 68.5 ± 1.7 d) because there was a tendency (P = 0.08) for a greater proportion of multiparous cows to be inseminated on estrus from 49 ± 3 to 62 ± 3 DIM (41.7 compared with 51.6%). Interval from calving to first AI was longer (P = 0.02) for cows with lesser BCS at 3 ± 3 DIM (lesser = 72.3 ± 2.0, moderate = 68.8 ± 1.6, and greater = 68.5 ± 2.0 d) and a smaller (P = 0.05) proportion of them were inseminated between 49 ± 3 and 62 ± 3 DIM (lesser = 25.7, moderate = 48.4, and greater = 53.6%). Although BCS loss from 3 ± 3 to 62 ± 3 DIM did not (P = 0.56) affect the interval to first AI, cows that experienced excessive loss of BCS tended (P = 0.07) to be less likely to be inseminated on estrus (excessive = 40.0 compared with moderate = 48.7%). Cows diagnosed as estrous cyclic at 49 ± 3 DIM had a shorter (P < 0.01) interval to first AI compared with those diagnosed as anovular (68.7 ± 1.7 vs. 71.0 ± 1.7 d), because a greater (P < 0.01) proportion of estrous cyclic cows were inseminated on estrus before 62 ± 3 DIM (57.6 compared with 32.1%). Interval from calving to first AI was not affected by morbidity, but a smaller (P = 0.04) proportion of cows diagnosed with at least one disease was inseminated on estrus from 49 ± 3 to 62 ± 3 DIM (Table 2).

100

Proportion not pregnant, %

130

CC CT TT

75

50

25

0 50

75

100

125

150

175

200

225

250

275

300

Days after parturition Fig. 1. Survival curves for the interval between calving and pregnancy for cows according to leptin genotype.

(excessive = 34.9 and moderate = 49.7%). Anovular cows had a reduced (P < 0.01) proportion of pregnancy after the first two AI (39.6 compared with 52.0%). Similarly, cows diagnosed with at least one disease event were less likely (P < 0.01) to become pregnant after the first two AI (Table 2).

3.6. Reproductive performance at 305 DIM 3.3. First postpartum AI Pregnancies per AI at first were not associated with leptin genotype (Table 1). Anovular cows at 49 ± 3 DIM had reduced (P < 0.01) P/AI at 31 ± 3 (24.9 compared with 38.7%) and 60 ± 3 (19.8 compared with 30.7%) d after first AI. Similarly, cows diagnosed with at least one disease event were less (P < 0.01) likely to be pregnant 31 ± 3 and 60 ± 3 d after AI (Table 2). The proportion of cows that lost pregnancy from 31 ± 3 to 60 ± 3 d after AI was not associated with leptin genotype (Table 1). Primiparous cows were less (P = 0.04) likely to lose their pregnancy compared with multiparous cows (12.8 compared with 26.1%). Cows not diagnosed with a disease event had a 60% reduction (P = 0.01) in the odds of losing their pregnancy than cows diagnosed with disease (Table 2). 3.4. Second postpartum AI Leptin genotype was not associated with P/AI at the second AI (Table 1). Cows that had moderate BCS loss in the first 62 ± 3 DIM were more likely (P = 0.04) to be become pregnant at the second AI (excessive = 18.6 and moderate = 30.0%). Similarly, healthy cows also had increased P/AI at the second AI compared with cows diagnosed with a disease event (Table 2). 3.5. First and second postpartum AI Leptin genotype was not associated with the proportion of pregnant cows after the first two postpartum AI (Table 1). Cows that lost a moderate amount of BCS in the first 62 ± 3 DIM had increased (P = 0.02) proportion of pregnancy compared with cows that lost excessive BCS

The proportion of cows pregnant by 305 DIM was not associated with leptin genotype (Table 1). Cows with lesser BCS at 62 ± 3 DIM (72.7 compared with 83.6%; P = 0.02) and cows that experienced excessive BCS loss from 3 ± 3 to 62 ± 3 DIM (61.1 compared with 80.2%; P < 0.01) were less likely to be pregnant by 305 DIM. There was a tendency (P = 0.06) for estrous cyclic status to influence pregnancy by 305 DIM, and more estrous cyclic than anovular cows were pregnant by 305 DIM (72.2 compared with 80.8%). Similarly, healthy cows were 2.1 times more likely (P < 0.001) to become pregnant within 305 DIM compared with cows diagnosed with disease (Table 2). Leptin genotype was not associated with the rate at which cows became pregnant (Table 3), which resulted in no differences in the interval to pregnancy (Fig. 1). Primiparous cows tended (P = 0.08) to have a greater pregnancy rate (AHR = 1.16; 95% CI = 0.98–1.38) than multiparous cows, which reduced the median days open by 14 d (129 compared with 143 d). Body condition score at 62 ± 3 DIM was also associated (P = 0.02) with pregnancy rate, and cows with moderate to greater BCS had a 22% greater rate of pregnancy (AHR = 1.22; 95% CI = 1.04–1.44) than cows with a lesser BCS. Similarly, cows that lost less than 1 unit of BCS from calving to 62 DIM had a 60% greater pregnancy rate (AHR = 1.60; 95% CI = 1.24–2.05) than those that lost 1 or more units of BCS. In addition to parity and BCS, morbidity and estrous cyclic status at 49 DIM also influenced (P < 0.001) pregnancy rate. Cows diagnosed with at least one disease event postpartum had a 36% decrease in the rate of pregnancy (AHR = 0.64; 95% CI = 0.52–0.78) compared with healthy cows, which extended the median interval to pregnancy by 60 d (Fig. 2). Cyclic cows on d 49 postpartum had a 35% increased pregnancy rate (AHR = 1.35; 95% CI = 1.14–1.61) compared with anovular

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Table 3 Interval to pregnancy in lactating Holstein cows according to leptin genotype. Genotypea

Cows

Censored (%)

CC CT TT

282 392 140

58 (20.6) 93 (23.7) 34 (24.3)

a b c

Interval to pregnancy, d Median

Mean ± SEM

132 129 158

156.2 ± 4.8 156.5 ± 4.2 163.3 ± 6.2

. Proportion not pregnant, %

1.11 1.21 Referent

95% CIc

0.87–1.41 0.89–1.41 –

P

0.61

Single nucleotide polymorphism in the R4C locus in the Exon-2 region of the leptin gene. AHR = adjusted hazard ratio. CI = confidence interval.

100

75 No Disease Disease

50

25

0 50

75

100

125

150

175

200

225

250

275

300

Days after parturition Fig. 2. Survival curves for the interval between calving and pregnancy according to morbidity. Compared with no disease cows, the adjusted hazard ratio for pregnancy rate in cows diagnosed with disease was 0.64 (95% CI = 0.52–0.78; P < 0.001). The median and mean (±SEM) days to pregnancy were, respectively, 122 and 150.0 ± 3.2 for no disease cows and 182 and 185.3 ± 5.7 for cows diagnosed with disease.

cows, thereby reducing the median interval to pregnancy from 156 to 122 d (Fig. 3). 4. Discussion The proportions of cows bearing C and T alleles in the present study were similar to that described by Buchanan 100

Proportion not pregnant, %

AHRb

75 Anovular Cyclic

50

25

0 50

75

100

125

150

175

200

225

250

275

300

Days after parturition Fig. 3. Survival curves for the interval between parturition and pregnancy according to estrous cyclic status at 49 ± 3 DIM. Anovular, cows with progesterone <1.0 ng/mL at 35 ± 3 and 49 ± 3 DIM; estrous cyclic, cows with at least one sample in which progesterone ≥1 ng/mL. Compared with anovular cows, the adjusted hazard ratio for pregnancy rate in estrous cyclic cows was 1.35 (95% CI = 1.14–1.61; P < 0.001). The median and mean (±SEM) intervals to pregnancy were, respectively, 122 and 148.8 ± 3.7 d for estrous cyclic cows and 156 and 171.8 ± 4.6 d for anovular cows.

et al. (2003). The presence of SNP in the R4C locus of the Exon-2 region of the leptin gene is thought to be important for the activity of the leptin hormone, because the change from a C to T nucleobase results in the presence of an unpaired cysteine and a change in the tertiary conformation of the leptin protein, which has been suggested to potentially affect biding to leptin receptor and alter the signaling pathway for the hormone action (Konfortov et al., 1999; Buchanan et al., 2002). Leptin resistance has been observed in obese rats and this is supposed to be mediated by changes in transcription factors downstream the leptin–leptin receptor binding caused by an unpaired cysteine amino acid mutation in the leptin gene (Zabeau et al., 2005). A smaller proportion of cows homozygous for the T allele was estrous cyclic by 49 DIM. Delayed resumption of postpartum ovulation in lactating dairy cows is associated with the state of energy balance, as dairy cows resume normal ovulatory cycles approximately 10 d after reaching the nadir of energy balance (Beam and Butler, 1999). Furthermore, cows that experienced ovulation earlier after parturition have greater concentrations of insulin and IGF-I, which are related to energy status and seem to be critical for the re-establishment of normal pulsatility of LH release, development and differentiation of follicles and their steroidogenic capability, and ovulation (Butler, 2000). Considering that BCS is an indirect measure of energy status, it is interesting to note that a smaller proportion of cows homozygous for the T allele were estrous cyclic at approximately 49 DIM, despite the fact that they had greater average BCS during the first 60 DIM compared with CT cows (Chebel et al., 2008). This is an indication that restoration of ovulation postpartum after the nadir of energy balance is reached could also be associated with leptin concentrations. Although leptin is one of the endocrine signals critical for normal fertility in mouse models (Barash et al., 1996), SNP of the coding region of the leptin gene was not associated with energy balance or resumption of ovulation in one study (Liefers et al., 2002). Also, in spite of polymorphisms in the coding region of the leptin gene having been associated with feed intake (Liefers et al., 2002) and milk yield (Liefers et al., 2002; Chebel et al., 2008) of lactating dairy cows, the association with earlier ovulation postpartum and subsequent fertility are not clear. Infertility of ob/ob mice, which are deficient in leptin, can be reversed by treatment with leptin, which results in increased secretion of LH and FSH, and increased ovarian weight (Barash et al., 1996). Leptin appears to have a positive feedback on the hypothalamus to stimulate secretion of GnRH, and has a positive feedback on the pituitary

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gland to stimulate secretion of LH and FSH in various mammals (Barash et al., 1996; Amstalden et al., 2002; Zieba et al., 2003). In Holstein cows, a prolonged decline in leptin concentration following parturition was associated with an extended interval to first postpartum ovulation (Kadokawa et al., 2000), and leptin concentrations tended to increase towards first ovulation (Liefers et al., 2003). Previous research has indicated that the concentrations of leptin before calving and shortly thereafter are less in cows with TT genotype than cows bearing CC or CT genotypes (Liefers et al., 2003), which could influence postpartum ovulation and explain why more cows homozygous for the T allele were anovular by 49 DIM in the present study. There was no association between leptin genotype and the proportion of anovular cows that resumed estrous cyclicity between 49 and 62 DIM or on the proportion of cows diagnosed cyclic by 62 DIM. It is possible that leptin genotype did not affect the proportion of cows resuming estrous cyclicity because differences in leptin concentration between cows homozygous for the C and T allele have only been observed before calving and shortly thereafter (Liefers et al., 2003). Also, 521 of the 814 cows in the study received a CIDR between 42 and 49 DIM, which stimulates resumption of ovulation in anovular cows (Cerri et al., 2009). This could have masked a potential association of leptin genotype on the ability of anovular cows to resume ovulation between 49 and 62 DIM. Nevertheless, before any hormonal intervention, the TT genotype was less likely to have estrous cyclic cows despite having increased mean BCS (Chebel et al., 2008). Even though TT cows were more likely to be anovular by 49 DIM, there was no difference in the average DIM at first postpartum AI or the proportion of cows inseminated on detected estrus according to leptin genotype. The submission of cows to a timed AI protocol probably limited the negative effects of delayed estrous cyclicity on the interval from calving to AI. Therefore, the reproductive management imposed might have masked potential differences in reproductive performance caused by delayed estrous cyclicity and insemination. Numerically, a smaller proportion of cows homozygous for the T allele was inseminated on estrus, but it is possible that the relatively small number of TT cows (n = 140) compared with CC and CT in the study population, representing only 17.2% of the population, might have limited the ability to detect statistical differences. Anovular cows typically are less likely to be observed in estrus and inseminated following treatment with PGF2␣ (Chebel et al., 2006), but differences in the prevalence of cyclic cows at 49 DIM among genotypes might not have been sufficient to influence detection of estrus in the 13 d after treatment with PGF2␣ . There were no differences in P/AI after first or second postpartum AI, in the proportion of cows pregnant by 305 DIM, and in pregnancy rate according to leptin genotypes. Although genotype was associated with resumption of ovulation by 49 DIM, cows of the three leptin genotypes had similar reproductive performance, which resulted in no differences in median and mean intervals from calving to pregnancy. Recently, Komisarek and Antkowiak (2007) evaluated 219 Jersey cows for SNP in the different loci of the leptin gene. No relationships between SNP in the R4C

region and measures of reproductive performance were observed; however, there was an association between SNP in the A59V locus of the leptin gene and reproduction. Cows bearing the TT genotype had shorter calving interval and fewer inseminations per pregnancy. Because cows not observed in estrus had time of ovulation synchronized, this strategy likely minimized a possible delay in first postpartum insemination. Previously, Chebel et al. (2008) observed that cows homozygous for the T allele were more likely to experience postpartum diseases (Chebel et al., 2008) and were less likely to be estrous cyclic at 49 DIM, which are conditions that clearly affect fertility. Even when the rate of pregnancy in the first 305 DIM was analyzed with only genotype in the model, no difference was observed and the AHR and 95% CI were 1.16 (0.92–1.46) for CC (P = 0.21) and 1.12 (0.90–1.40) for CT (P = 0.31) compared with TT cows, respectively. Therefore, the lack of a genotype influence on reproductive performance of lactating Holstein cows is unlikely to have been masked by differences in estrous cyclic status or risk of postpartum diseases in the statistical analyses. Considering the proportion of TT cows diagnosed as anovular at 49 DIM and the P/AI of anovular cows in the current study, the expected reduction in P/AI after first AI for TT cows because of anovular condition would be 1.5 percentage units compared with CC and CT cows. Similarly, considering the proportion of TT cows that experienced a disease event and the P/AI of cows with disease, the expected reduction in P/AI for TT cows because of disease should be 0.9 percentage unit compared with CC and CT cows. This corresponds to an expected reduction in P/AI after first postpartum insemination of 2.4 percentage units for TT cows, which is not much different than the numerical differences in P/A observed in the present study. Therefore, although cows homozygous for the T allele have increased incidence of anovular condition and disease (Chebel et al., 2008), which would make them less fertile, it seems these effects are small, which resulted in similar reproductive performance in the population of cows studied and managed under an intensive reproductive management system. Cows with postparturient diseases were less likely to be estrous cyclic at 49 and 62 DIM, and anovular cows at 49 DIM that experienced disease were less likely to resume estrous cyclicity by 62 DIM. Disease had striking effects suppressing fertility of dairy cows, ultimately resulting in a 53% reduction in the odds of a cow to be pregnant by 305 DIM, a 36% reduction in the rate of pregnancy, and a 60 d increase in median days open. Occurrence of diseases such as retained fetal membranes (Chebel et al., 2004), mastitis (Santos et al., 2004; Chebel et al., 2004), and lameness (Garbarino et al., 2004; Hernandez et al., 2005) have been associated with reduced fertility in lactating dairy cows. In fact, clinical diseases diagnosed in the first 60 DIM reduced the prevalence of lactating dairy cows ovulating in the first 65 d in lactation, compromised P/AI at first postpartum insemination, and increased the risk of pregnancy loss in the first 60 d of gestation (Santos et al., 2011). The exact mechanisms by which morbidity early in lactation influences reproductive performance of dairy cows are not clearly defined and, likely, multifactorial. Neverthe-

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less, cows diagnosed with diseases usually have reduced appetite and lose more BCS. Furthermore, in the current study, cows diagnosed with a disease event experienced delayed resumption of ovulation at the different DIM evaluated. Reduced BCS, increased loss of BCS, and anovular condition are all risk factors associated with suppression of fertility in lactating dairy cows (Santos et al., 2009). Interestingly, the proportion of cows with CC, CT, and TT genotype was not what is expected according to Mendelian inheritance, in which the proportion of cows with CC and TT genotypes should be approximately 25% and those with CT genotype approximately 50%. In the present study, only 17% of cows had TT genotype, even though these cows produced more milk (Chebel et al., 2008). This could be a consequence of the fact that TT cows were more likely to experience disease (Chebel et al., 2008) and be anovular, which may impact their fertility, making them more likely to be removed from the herd population. Although heterozygosis for the leptin gene might confer improvements in lactation performance (Liefers et al., 2002; Chebel et al., 2008) and some disease resistance (Chebel et al., 2008), the current study was not able to demonstrate similar improvements in reproductive performance according to SNP in the coding region of the leptin gene when cows were subjected to a controlled reproductive management program. 5. Conclusions Leptin genotype has been associated with productive responses of Holstein cows, but reports on the association between SNP for the leptin gene and reproductive performance of high producing Holstein cows are scarce. Cows homozygous for the T allele were more likely to be anovular at 49 DIM, despite having greater average BCS in early lactation than CT cows. Despite differences in the prevalence of estrous cyclic cows before the end of the voluntary waiting period, SNP for the leptin gene did not confer additional benefits to fertility of dairy cows as measured by detection of estrus, P/AI at the first and second postpartum AI, and maintenance of pregnancy following the first postpartum AI. The lack of association between genotype and fertility responses resulted in absence of differences in the rate of pregnancy and interval from calving to pregnancy in lactating Holstein cows according to SNP in the leptin gene. Morbidity in early lactation had remarkable impacts on measures of fertility and on reproductive performance of dairy cows, and cows diagnosed with at least one disease event had a 60 d longer median interval to pregnancy. These results do not support a beneficial impact of selecting lactating dairy cows to alter the distribution of SNP of the leptin gene in the cattle population to improve reproductive performance. Nevertheless, these results reinforce the concept that managerial practices that reduce the risk for morbidity are critical for good reproductive performance of Holstein cows. References Amstalden, M., Garcia, M.R., Stanko, R.L., Nizielski, S.E., Morrison, C.D., Keisler, D.H., Williams, G.L., 2002. Central infusion of recombinant

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