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Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

We aimed to evaluate the treatment with estradiol benzoate (EB) or 17β-estradiol (E2) associated with progesterone (P4) for resynchronization of ovulation 14 days after timed artificial insemination (TAI). In Experiment 1 (Exp. 1), Nelore heifers were submitted to TAI (D0). On D14, the animals received an intravaginal P4 device and were randomly assigned to one of three groups: control (no treatment; n = 17); EB (1 mg EB; n = 17); and E2+P4 (1 mg E2 + 9 mg P4; n = 18). Ultrasonography evaluations were performed daily from D14 to D22 to map follicular and luteal dynamics. On D22, the P4 devices were removed and non-pregnant (NP) animals were determined using corpus luteum blood flow Doppler ultrasonography. In Exp. 2, 1295 beef heifers were resynchronized and randomly allocated to the same experimental groups as described in Exp. 1. On D22, the largest follicle (LF) was measured in NP and a second TAI was performed on D24. In a subset of heifers (n = 337), an estrus detection patch was used between D22 and D24 to monitor estrus expression and the LF was measured at D24. Confirmatory diagnosis of pregnancy was performed between D37-67 and D43-67 after first and second TAI, respectively. In Exp 1, the proportion of heifers with a synchronized follicular wave emergence (from 3 to 5 days after treatment) was greater (P 0.05) in the EB group (93.8%) than in the control (62.5%) and E2+P4 (64.7%) groups. Structural luteolysis occurred earlier (P 0.05) in the EB and E2+P4 groups than in the controls. The pregnancy rate after first TAI did not differ (P 0.1) among the groups at D22 and at confirmatory diagnosis in both experiments. In Exp 2, the potential pregnancy loss between D22 and D37-67 was similar (P 0.1) in the control (19% [36/185]), EB (15% [28/182]) and E2+P4 (15% [28/184]) groups. The LF diameter (mm) on D22 was greater (P 0.05) in the control group (11.9 ± 0.1) than in EB (11.3 ± 0.1) and E2+P4 (11.5 ± 0.1). No difference (P 0.1) was observed in the proportion of heifers detected in estrus, but LF growth rate (mm/day) between D22 and D24 was greater (P 0.05) in EB group (0.9 ± 0.08) than in control (0.6 ± 0.07) and E2+P4 (0.7 ± 0.09) groups. The pregnancy rate for the second TAI was greater (P 0.05) in the EB group (47% [94/200]) than in the control (37% [76/203]), but did not differ (P 0.1) from the E2+P4 group (43% [93/214]). In conclusion, the treatment with 1 mg EB or 1 mg E2 + 9 mg P4 at 14 days post-TAI anticipates luteolysis in NP heifers but does not compromise pregnancy. The EB treatment induces a new synchronized follicle wave emergence and increases the pregnancy rate of resynchronized NP heifers.

We aimed to evaluate the reproductive performance of Nelore lactating cows submitted to a resynchronization 12 days after timed artificial insemination (TAI) with or without a long-acting progesterone (P4-LA) treatment. Nelore cows were submitted to a P4/oestradiol-based TAI protocol (D0 = insemination). On D12, cows in the control group (n = 184) received a new P4 intravaginal device (0.96 g), whereas cows in the P4-LA group (n = 192) received the P4 device and 75 mg P4-LA. Cows identified as non-pregnant (n = 120) by regression of corpus luteum using colour Doppler ultrasonography on D20 had the P4 device removed and received 500ug of sodium cloprostenol, 1 mg of oestradiol cypionate and 300 IU of eCG and were re-inseminated on D22. There was no difference (p 0.10) in the pregnancy rate at D20, D30 and D60 after first TAI between the control (69%, 59.7% and 57%, respectively) and P4-LA (67%, 55.7%, and 55.2%, respectively) groups. Pregnancy losses were similar between both groups (p 0.1). For cows submitted to the second TAI, the pre-ovulatory follicle size did not differ (p 0.1), but the oestrous detection and pregnancy rates were greater (p 0.05) in the P4-LA group (92.2% [59/64] and 60.9% [39/64], respectively) than in controls (75% [42/56] and 44.6% [25/56]). The cumulative pregnancy rate after two TAIs did not differ (p 0.1) between control (73.3% [135/184]) and P4-LA (76% [146/192]) groups. The use of P4-LA at 12 days after TAI potentially increases the pregnancy rates for a new early resynchronization strategy associated with the Doppler imaging for pregnancy diagnosis and results in an alternative to perform two TAIs in 22 days in beef cows.

Different approaches have been used to improve conception rates at foal heat. Omega-3 fatty acids family and derivatives have improved reproductive efficiency in ruminants, but literature lacks studies evaluating these components on equines. The objective of this study was to evaluate effects of mare dietary supplementation with microalgae rich in docosahexaenoic acid (DHA) during peripartum on follicular dynamics and uterine involution in early post-partum. Eighteen pregnant mares, no particular breed, 410 ± 39.5 kg body weight (BW), and 7.83 ± 2.01 yr old were used. Mares were randomly assigned to control (CONT) or supplementation with microalgae rich in DHA at 0.06 g/kg BW (ALG). Treatments were supplied from 90 d prior to expected foaling date until 7 d after first ovulation. Reproductive evaluations were performed during early post-partum until 7 d after first ovulation through rectal palpation and ultrasonography of the following parameters: uterine and endometrium diameters, intrauterine fluid (IUF), uterine echogenicity, uterus tone, and follicular dynamics. Endometrial cells samples were collected to assess mRNA expression of CRP, IL-1β and AKR1C4, using RT-qPCR. Data were analyzed by mixed procedure of SAS. ALG mares had smaller uterine horns diameters and greater uterine echogenicity during post-partum in comparison with CONT. No treatment effects were detected for other characteristics evaluated, but a day effect was observed for uterine and endometrium diameter, IUF, uterine echogenicity, and transcript abundance of endometrial AKR1C4. Supplementation with DHA during peripartum may benefit uterine involution process and odds of early conception, but more studies should be performed regarding fertility.

To determine effects of delaying the injection of prostaglandin F2α (PGF) and fixed-time artificial insemination (TAI) in the 14-d CIDR-PG protocol, 1,049 Angus heifers at six locations were enrolled in a completely randomized design. Within location heifers were randomly assigned to one of two treatment groups: 1) PG16 (n = 518), heifers received a controlled internal drug release (CIDR) insert on d 0 for 14 d, a 25-mg injection of PGF 16 d after CIDR removal (d 30), and a 100-µg injection of gonadotropin-releasing hormone concurrent with TAI 66 ± 2 h later; or 2) PG17 (n = 531), heifers were treated the same as PG16, however, PGF was administered 17 d after CIDR removal (d 31), and heifers were TAI 66 ± 2 h later. Estrus detection patches were applied to a subset (n = 482) of heifers at the time of PGF administration and were examined for activation at TAI. Dominant follicle diameter was determined via transrectal ultrasonography at PGF administration and TAI in a subset of heifers (n = 116). Transrectal ultrasonography was performed to determine pregnancy rates to TAI (PR/AI) between 30 and 45 d after TAI. Estrus expression prior to TAI differed by treatment where PG17 heifers had greater (P < 0.01) expression of estrus than PG16 heifers (57.8 ± 6.1% vs. 43.4 ± 6.1%, respectively). Nevertheless, dominant follicle diameters at PGF and at TAI were similar (P ≥ 0.59) between PG16 and PG17 heifers. In addition, PR/AI did not differ (P = 0.29) between PG16 and PG17 treatments (50.5 ± 3.2% vs. 45.7 ± 3.1%, respectively). Results of this experiment indicate that delaying the injection of PGF and TAI in the 14-d CIDR-PG protocol increased estrus expression prior to TAI yet did not improve fertility in beef heifers.

The objective of this study was to evaluate the use of corpus luteum (CL) color doppler ultrasonography (CD) for early pregnancy diagnosis in Bos taurus beef cows and evaluate differences in CL blood perfusion among cows that experience late embryonic/early fetal mortality (LEM). Postpartum Bos taurus beef cows (n = 214) from three locations were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Cows were considered non-pregnant when CL area was < 20 mm2 or estimated blood perfusion was ≤ 25%. Conventional ultrasonography on day 29 was utilized as the gold standard method. Pregnant cows had greater CL diameter, CL area, and CL volume when compared to non-pregnant cows on day 20 and 22 (P < 0.001; Table 1). Additionally, percentage of central, peripheral, and total luteal blood perfusion, as well as the respective blood perfusion scores were greater (P < 0.001) in pregnant compared with non-pregnant cows on both day 20 and 22. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy for the CD on day 20 were 100, 74, 81, 100, and 88%, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for CD on day 22 were 100, 86, 89, 100, and 93%, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated no differences between CD on days 20 and 22 (P = 0.12). No differences in luteal blood perfusion were observed on day 20 or 22 among cows that maintained pregnancies compared with LEM cows (P ≥ 0.27). In conclusion, CD successfully detected most non-pregnant cows (81 and 89% on day 20 and 22, respectively) and false negative results were absent (NPV = 100%).

The intensive use of Doppler ultrasonography in several studies in the last decade allowed the characterization of vascular perfusion and the estimation of function of the reproductive organs and tissues along the estrous cycle and pregnancy in cattle. We aim to discuss the possibility of using Doppler imaging and to explore the potential of its inclusion in reproductive programs in cattle industry. Recent studies in dairy and beef cows indicated a high accuracy and sensitivity when Doppler ultrasonography is used to evaluate corpus luteum function and to diagnosis pregnancy between days 20 and 22. Moreover, resynchronization programs starting 5 to 7 days after timed embryo transfer (FTET) coupled with early pregnancy diagnosis were developed for beef cattle, and have been implemented in commercial embryo transfer programs. These strategies allow a reduction in the interval between two FTET from ≈ 40 to 24 days and may improve the gains in reproductive efficiency when compared to traditional programs than begin resynchronization after the pregnancy diagnosis at 30 days. A second alternative to use Doppler imaging is the evaluation of luteal blood perfusion at the time of embryo transfer for selection of recipients with greater receptivity potential. This may increases fertility in FTET, as embryos would not be transferred to females with non-functional CL, and in cases with recipients surplus, females with higher receptivity would be prioritized.

The objective of this study was to evaluate the use of plasma concentrations of pregnancy associated glycoproteins (PAG) for pregnancy diagnosis in beef cows on day 25 of gestation. Additionally, differences in circulating concentrations of PAG among cows that experienced late embryonic/early fetal mortality (LEM) were evaluated. Beef cows (n = 214) were exposed to the 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). Blood samples were collected on days 25 and 29 to determine circulating concentrations of PAG and ultrasonography on day 29 was used as the gold-standard method for pregnancy diagnosis. On days 25 and 29, PAG concentrations were greater (P < 0.001) in pregnant compared with non-pregnant cows. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy on day 25 were 87, 80, 83, 84, and 83%, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for PAG on day 29 were 99, 94, 96, 97, and 99%, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated greater area under the curve for PAG on day 29 compared with day 25 (P < 0.001). There were no differences in plasma concentrations of PAG on day 25 among cows that experienced LEM compared to cows that maintained pregnancy (P = 0.46). Concentrations of PAG on day 29 were reduced (P = 0.03) in cows that experienced LEM compared with cows that maintained pregnancy, and were not different between LEM and non-pregnant cows (P = 0.12). In summary, cows that experienced LEM had decreased plasma concentrations of PAG compared with cows that maintained pregnancy. The PAG assay utilized in this study yielded accurate pregnancy diagnosis results on day 29 of gestation: however, the presence of false negative results (NPV = 84%) limits its use on day 25 of gestation.

To determine the effects of delaying the injection of prostaglandin F2α (PGF) and fixed-time artificial insemination (TAI) in the 14-d CIDR-PG & TAI protocol, 911 Angus heifers at 5 locations were enrolled in a completely randomized design. Within location heifers were randomly assigned to 1 of 2 treatment groups: 1) PG16 (n = 452), heifers received a CIDR insert on d 0 for 14 d, a 25-mg injection of PGF 16 d after CIDR removal [d 30], and a 100-µg injection of gonadotropin-releasing hormone concurrently with TAI 66 ± 2 h later; or 2) PG17 (n = 459), heifers were treated the same as PG16, however, PGF was administered 17 d after CIDR removal [d 31], and heifers were TAI 66 ± 2 h later. Estrus detection patches were applied to heifers at the time of PGF administration and were examined for activation at TAI. Dominant follicle diameter was determined via transrectal ultrasonography at PGF administration and TAI in a subset of heifers (n = 171). Furthermore, transrectal ultrasonography was performed to determine pregnancy rates to TAI (PR/AI) between 30 and 45 d after TAI. Estrus expression prior to TAI was similar (P = 0.50) between treatments (48.9 vs. 52.0%, respectively). Moreover, dominant follicle diameter at PGF and TAI was similar (P ≥ 0.43) between PG16 and PG17 heifers. Pregnancy rates to TAI did not differ (P = 0.48) between treatment groups (46.4 vs. 48.9%, respectively). The results of this experiment indicate that delaying the injection of PGF from d 30 to d 31 along with TAI in the 14-day CIDR-PG & TAI protocol had no effects on fertility parameters in beef heifers. In conclusion, the PGF injection and TAI in the 14-d CIDR-PG & TAI protocol may be delayed, providing more flexibility in scheduling without negatively affecting fertility.

Made available in DSpace on 2021-06-25T11:07:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T15:24:05Z : No. of bitstreams: 1 S1984-31432020000300505.pdf: 760850 bytes, checksum: 3028cce735a7e1fa219c5892e4143b7c (MD5) National Institute of Food and Agriculture Reproductive failure and pregnancy loss in cattle are some of the largest economic burdens to cattle producers and one of most perplexing factors influencing management decisions. Pregnancy loss may occur at any point during gestation with the largest percentage of loss occurring in the first 30 days and, subsequently, decreasing as the pregnancy progresses. Losses may be attributed to numerous factors, predisposed issues or environmental conditions such as nutritional stressors or disease. From a research perspective, determining the exact causes of pregnancy loss or embryonic mortality in cattle have been difficult, due to limitations of accurately determining early gestation pregnancy status. Until methods that precisely determine embryo success early in gestation are available, our understanding of in vivo pregnancy loss will lack clarity necessary to develop management strategies to decrease such loss. In this review, we will briefly discuss the pivotal periods of pregnancy loss affecting beef and dairy cattle, methods and technologies to determine pregnancy status and embryo viability and potential opportunities to decrease reproductive failure. Department of Animal Science Pregnancy and Developmental Programming Area of Excellence Texas A&M University College Station Departamento de Produção Animal Universidade Estadual Paulista (UNESP) Departamento de Produção Animal Universidade Estadual Paulista (UNESP) National Institute of Food and Agriculture: W3112-TEX07702

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P 0.05) from the PL group. ISG15 expression was greater (P 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

The objective of this experiment was to compare the expression of Interferon-tau Stimulated Genes (ISGs) in peripheral blood mono and polymorphonuclear cells (PBMCs and PMNs) in heifers following insemination. Twenty-nine Nelore heifers had estrous cycle synchronized, and FTAI occurred on D0. Pregnancy diagnosis was performed by ultrasonography on D28 post FTAI. On D0, 10, 14, 16, 18 and 20, blood (25mL) from the jugular vein was collected in heparinized tubes for isolation of PMNs and PBMCs. The isolation was performed using Ficoll®Paque Plus (GE Healthcare). PMNs and PBMCs samples from 8 pregnant and 9 non-pregnant heifers were subjected to RNA extraction using the DirectZol-RNA kit (Zymo-Research) and Trizol (Invitrogen), respectively. The expression of the target genes (ISG15, OAS-1, MX1 and MX2) was normalized in relation to the two reference genes (GAPDH/ACTB for PMNs and GAPDH/PPIA for PBMCs). The abundance of transcripts was evaluated by analysis of variance considering fixed effects of group, day and group by day interaction using the PROC MIXED procedure in SAS. PMNs and PBMCs had a similar expression profile of ISG15 and OAS-1, showing a relative increase (P < 0.05) from D18, and a significant increase in (P < 0.05) expression in pregnant compared to non-pregnant females on D18 and D20. These results were also observed for MX1 in PBMCs. In PMNs, no significant effects for MX1 were found. For MX2, in both cells types, only a group effect (P < 0.05) was observed, indicating a higher expression in pregnant heifers (0.57±0.11 vs. 0.21±0.03) on the days evaluated. When comparing the relative expression of the target genes to D0, no significant (P > 0.1) differences were found between PMNs and PBMCs. In summary, ISG expression is similar in PMNs and PBMCs, specifically for ISG15 and OAS-1, which both seem to be suitable biomarkers for potential early pregnancy determination in heifers.

We aimed to study the association between CL characteristics assessed by color-Doppler ultrasonography (Doppler-US) at the time of embryo transfer (ET) and pregnancy rate (P/ET) in beef recipients. Estrous cycles of crossbred beef recipients were synchronized for timed-ET. On the day of ET (Day 7), CL area, proportion of luteal blood perfusion (BP), and the relationship between the largest dominant follicle (DF) and CL (ipsilateral or contralateral) were determined. Animals (n = 444) received an in vitro produced embryo from Nelore donors, placed in the uterine horn ipsilateral to the CL. Recipients were split retrospectively in three subgroups according to CL area [small ( 4 cm2)] and three subgroups according to luteal signals of BP [low (≤40%), medium (45–50%) or high (≥55%)]. Pregnancy was detected on Days 30–45 by transrectal ultrasonography and P/ET was analyzed considering the effects of cow's category (suckling or non-suckling), CL area, luteal BP and side of DF. P/ET increased along with BP category [low, 45.9%B, (62/135); medium, 54.1%AB (93/172); and high, 58.4%A (80/137)]. When luteal BP was evaluated as a continuous variable, a significant (P 0.1], when CL area was evaluated as a continuous variable, a quadratic effect (P 0.1). In conclusion, Doppler-US is an innovative tool that has the potential to be used for selection of suitable embryo recipients based on luteal BP. Selection of recipients that have a greater chance of maintaining pregnancy will increase the success of timed-ET programs.

We aimed to compare three methods to detect pregnancy based on interferon-tau stimulated gene expression in peripheral blood polymorphonuclear cells and Doppler ultrasonography (Doppler-US) 20 days after timed AI and the concentrations of pregnancy associated glycoproteins (PAGs) 25 days after timed AI. Our second objective was to compare interferon-tau stimulated genes and PAGs as early embryo loss (EEL) predictors. Nelore cows (n=144) and heifers (n=103) were submitted to timed AI (Day (D) 0). On D20, polymorphonuclear cells were isolated from blood samples by Ficoll gradient (GE Healthcare), and the RNA was extracted. Expression of the interferon-tau stimulated gene (OAS-1) was quantified by qPCR and normalized to reference genes (GAPDH and ACTB). On D25, blood was collected from the jugular vein and PAG concentrations were measured by an in-house enzyme-linked immunosorbent assay. Pregnancy diagnoses were performed on D20 by luteolysis detection with Doppler-US and on D30 by detection of an embryo by heartbeat. Animals were classified as pregnant (P; fetus on D30), non-P (NP; no active corpus luteum on D20), and EEL (active corpus luteum on D20 but NP on D30). Expression of OAS-1 and PAG concentrations were analysed by analysis of variance using PROC MIXED of SAS considering the effects of group, category, and their interaction. Receiver operating characteristic curves were created, and the area under the curve (AUC), accuracy, specificity, and sensitivity were calculated for pregnancy predictions on D20 (OAS-1 and Doppler-US) and D25 (PAGs) compared with the standard diagnosis method on D30. Expression of OAS-1 and PAG concentrations were greater (P

This study characterised the expression of interferon (IFN)-τ-stimulated genes (ISGs) and Type I IFN receptors in circulating polymorphonuclear cells (PMNs) of beef heifers and compared it with expression in peripheral blood mononuclear cells (PBMCs) up to Day 20 of gestation. Nelore heifers (n=26) were subjected to fixed-time AI (FTAI) on Day 0. PMNs and PBMCs were isolated on Days 0, 10, 14, 16, 18 and 20 after FTAI. The abundance of target transcripts (ubiquitin-like protein (ISG15), 2′-5′-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (MX1), myxovirus resistance 2 (MX2), IFN receptor I (IFNAR1) and IFN receptor 2 (IFNAR2)) was determined using real-time quantitative polymerase chain reaction and compared between pregnant (n=8) and non-pregnant (n=9) females. In both PBMCs and PMNs, ISG15 and OAS1 expression was greater in pregnant than non-pregnant heifers on Days 18 and 20. There were no significant differences in the expression of ISGs between PBMCs and PMNs. A time effect on expression was found for IFNAR1 in PBMCs and IFNAR2 in PMNs, with decreased expression of both genes on Days 18 and 20. When the expression of these genes was compared between cell types only in pregnant heifers, IFNAR2 expression in PMNs had an earlier decrease when compared to its expression in PBMCs, starting from Day 18. In conclusion, PMNs do not respond earlier to the conceptus stimulus, and ISG15 and OAS1 expression in both PMNs and PBMCs can be used as a suitable marker for pregnancy diagnosis on Days 18 and 20. In addition, gestational status did not affect IFNAR1 and IFNAR2 expression, but IFNAR2 showed a distinct response between PMNs and PBMCs of pregnant heifers.