Low Incidence of an Altered Endometrial Epidermal Growth Factor (EGF) Profile in Repeat Breeder Holstein Heifers and Differential Effect of Parity on the EGF Profile Between Fertile Holstein (Dairy) and Japanese Black (Beef) Cattle

A high incidence (about 70%) of alteration in endometrial epidermal growth factor (EGF) profile, i.e., loss of 2 peaks on days 2–4 and 13–14, has been linked to a reduced fertility in multiparous repeat breeder Holstein cows. However, the EGF profile in Holstein heifers and other breeds (types) of cattle has not been investigated. In study 1, EGF concentrations were determined using endometrial tissues obtained by biopsy on days 3, 7 and 14 from 84 fertile Holstein heifers to obtain a normal range and 53 repeat breeder Holstein heifers to estimate incidence of alterations in the EGF profile. In repeat breeder heifers, EGF concentrations were similar to fertile controls on 3 days and five animals (9.4%) had an altered EGF profile with EGF concentrations below the normal range on days 3 and 14. In study 2, EGF concentrations on day 3 were repeatedly examined from the nulliparous period to the third postpartum period in 28 Holstein (dairy) and 47 Japanese Black (beef) cattle. The effect of parity on EGF concentrations on day 3 was different between Holstein and Japanese Black cattle. In Japanese Black cows, the EGF concentrations were consistently high throughout the study period, while in Holstein cows, the EGF concentrations decreased after the second calving. In conclusion, unlike multiparous repeat breeder Holstein cows, an altered EGF profile may not be a major cause of repeat breeding in Holstein heifers, and the peak EGF concentrations around day 3 may decrease even in fertile populations of multiparous dairy cows, but not in beef cows.     

Leptin receptor expression and its change in association with the normalization of EGF profile after seminal plasma treatment in repeat breeder dairy cows

Factors associated with high milk production levels have been linked to alterations in the endometrial epidermal growth factor (EGF) profile, a cause of reduced fertility in dairy cows. Therefore, we examined the leptin system that connects nutritional status and reproduction in dairy cattle related to reduced fertility in repeat breeder cows. Plasma leptin concentrations were measured in 18 heifers, 20 high-yielding control cows, and 26 repeat breeder cows, showing an altered EGF profile. Then, all repeat breeder cows were infused with seminal plasma (SP) into the vagina at the next estrus to normalize the EGF profile, while heifers and control cows were infused with vehicle alone. All animals were examined for EGF profiles. Eighteen repeat breeder cows, nine heifers, and nine control cows were also determined for leptin receptor (Ob-R) expression levels in the estrous cycle before and after the infusion. SP normalized the EGF profile in 53.8% of the repeat breeder cows. Leptin concentrations were similar in all groups, regardless of the treatment results for the EGF profile. In contrast, Ob-R levels in repeat breeder and control cows were similar and higher than those in heifers before SP treatment. Ob-R in repeat breeders showing a normal EGF profile after treatment decreased to an intermediate level between heifers and control cows and may provide a clue to take measures against repeat breeding in dairy cows.     

A progestin-based treatment with a high dose of estradiol benzoate normalizes cyclic changes in endometrial EGF concentrations and restores fertility in repeat breeder cows

The objective of the present study was to examine the efficacy of a progestin-based treatment with a high dose of estradiol benzoate (EB) to normalize the epidermal growth factor (EGF) profile in the uterine endometrium and restore fertility in repeat breeder cows. Repeat breeder cows without peaks in their endometrial EGF concentrations on Days 3 and 14 of the estrous cycle were used throughout the study. The effect of 1 (standard dose), 2.5 and 5 mg of EB in a progestin-based treatment protocol (EB1, EB2.5 and EB5 treatments, respectively; n=5 for each group) on endometrial EGF concentrations was first examined. The EB1 and EB2.5 treatments in the repeat breeder cows produced a suppressed response in endometrial EGF compared with EB1 treatment in the fertile controls (n=5) and failed to restore the normal EGF profile during the next estrous cycle. However, EB5 treatment produced an increase in EGF concentrations similar to the fertile controls and normalized the endometrial EGF profile. The effects of the EB1 and EB5 treatments (n=30 for each group) on the endometrial EGF profile and fertility were then examined in the repeat breeder cows. The proportion of cows, with an EGF profile normalized by the treatments was higher in the EB5 group (66.7%) than in the EB1 (30.0%) and untreated control (13.3%; n=30) groups (P<0.01). The pregnancy rates of the cows having a normal EGF profile after treatment in the EB1 and EB5 groups were similar (88.9 and 85.0%, respectively) and higher than those of the cows having an abnormal profile within the same groups (19.0 and 30.0%, respectively, P<0.01). In summary, the endometrial response to EB in terms of the EGF concentration was suppressed in repeat breeder cows. A high dose (5 mg) of EB in a progestin-based treatment was found to be effective for restoration of a normal EGF profile and fertility in repeat breeder cows having lesser endometrial EGF concentrations on Days 3 and 14.     

Nutritional factors that regulate ovulation of the dominant follicle during the first follicular wave postpartum in high-producing dairy cows

During recent decades, milk production per cow has increased drastically due to improved management, nutrition, and genetic selection; however, the reproductive performance of high-producing dairy cows has been declining. One of the factors responsible for this low reproductive performance is negative energy balance (NEB). NEB affects the onset of first ovulation in early postpartum cows. It is generally accepted that early first ovulation positively relates to the resumption of normal ovarian function, first service, and conception rate in dairy cows. Hence, delayed first ovulation has a negative impact on subsequent fertility. The metabolic condition of cows in NEB shifts to catabolic metabolism, which in turn causes increased plasma growth hormone and non-esterified fatty acid concentrations and decreased plasma insulin-like growth factor-1, insulin, and glucose concentrations. On the other hand, plasma β-carotene concentrations decrease throughout the dry period and reach their nadir in about the first week postpartum, and this change reflects energy balance during the peripartum period. β-Carotene plays a role independently of vitamin A in the reproductive performance of dairy cows, and the positive relationship between supplemental β-carotene and reproductive function has been demonstrated in many studies during the past decades. However, β-carotene content in corn silage, which is a popular main feed in high-producing dairy cows, is very low. This review describes nutritional factors related to ovulation during the first follicular wave postpartum in dairy cows.     

Relationships between insulin-like growth factor-I,milk yield,body condition score,and postpartum luteal activity in high-producing dairy cows

The relations between body condition score (BCS), milk yield, serum insulin-like growth factor-I (IGF-I) profile, and luteal activity were investigated in postpartum dairy cows. Seventy-one healthy high-producing multiparous Holstein cows were subjected to transrectal ultrasound scanning twice weekly from the first to the eighth week postpartum. Blood samples were collected twice weekly to measure serum progesterone (P4) and every 2 weeks to detect serum IGF-I concentrations. BCS was monitored weekly after calving. Cows with serum P4 concentrations ≥1 ng/ml on at least two consecutive samplings were considered to have commenced luteal activity. Commencement of luteal activity (C-LA) was observed earlier than 45 days postpartum in 71.8% of cows while 28.2% showed C-LA later than 45 days. Prolonged luteal phase was the most common abnormal pattern of luteal activity observed. Cows with a C-LA earlier than 45 days postpartum had higher (P ≤ 0.05) mean serum concentrations of IGF-I than those with later C-LA. In addition, cows which showed C-LA earlier than 45 days postpartum had more optimal productive indices including shorter calving to conception interval and calving to first service interval (P ≤ 0.05), and fewer services per conception (P = 0.07). C-LA was significantly later in cows that lost more than 0.5 BCS units within 3 weeks postpartum than in those that lost less than 0.5 units BCS during the same interval (P = 0.02). We conclude that high-producing dairy cows with higher postpartum serum IGF-I concentrations have earlier commencement and normal luteal activity, and better reproductive performance. Severity and duration of BCS loss adversely affect commencement of luteal activity.     

Les protéines totales et leurs fractions dans le sérum et dans les sécrétions endométriales chez la vache atteinte d'infertilité sine materia

Total proteins, albumine and globulins of serum and uterine secretions were evaluated in two groups of 15 normal and 15 repeat breeder cows. Cyclic significant variations of all studied parameters were observed in uterine secretions of normal cows although only total proteins showed the same pattern in repeat breeder cows where individual variations were noted to be very important. Concentrations of total proteins, albumin (P<0.05) and β globulins (P<0.01) were different between the two groups during either the estrous or the postestrous period. Concentration of proteins in uterine fluid was higher in normal breeder cows during postestrus and diestrus.     

Effect of reproductive disorders and parity on repeat breeder status and culling of dairy cows in Quebec

This study quantified the effect of peripartum reproductive disorders and parity on repeat breeder status and involuntary culling of dairy cows. Reproductive data of 418 383 lactations were taken from a computerized databank of health records for dairy cows. A logistic regression model was used with dystocia, retained placenta (RP), metritis complex, and parity as fixed effect risk factors and herd entered as the random effect. Of the peripartum problems studied, dystocia had the greatest effect on future fertility. Dystocia increased the odds of a cow being a repeat breeder by 44% [odds ratio (OR): 1.44; confidence interval (CI): 1.37 to 1.51]. Compared to first-parity cows, cows in second, third, and fourth parities had significantly higher odds of being a repeat breeder: 18% (OR: 1.18; CI: 1.16 to 1.20); 24% (OR: 1.24; CI: 1.21 to 1.26); and 42% (OR: 1.42; CI: 1.39 to 1.45), respectively. The odds for second-, third-, or fourth-parity repeat breeders being culled were 24% (OR: 1.24; CI: 1.20 to 1.28); 39% (OR: 1.39; CI: 1.35 to 1.43); and 67% (OR: 1.67; CI: 1.62 to 1.71) respectively, while peripartum reproductive problems had less of an effect.     

持久黄体性不孕奶牛用复方缩宫素乳剂处理后血清中孕酮和雌二醇水平的变化

选择患持久黄体的不孕奶牛14头进行复方缩宫素乳剂的临床治疗试验,并利用放射免疫方法对部分患牛治疗前后血清孕酮（P     

Comparative biochemical profiles,production and reproduction status of the post‐partum dairy cows with and without purulent vaginal discharge

Purulent vaginal discharge (PVD) is a prevalent uterine disease of dairy cows during the puerperium that affects the milk production and affects the profitability of farms. The objectives of this study were to evaluate the biochemical profile, the body condition score, the milk production of cows with PVD and the effects PVD on reproductive performance. A total of 338 Holstein dairy cows aged from 3 to 5 years, from three commercial dairy farms, from Brazil, were used. Blood samples were collected within 25 ± 3 days post‐partum from Holstein dairy cows without PVD (control cows, n = 242) and cows with PVD (n = 96), based on scoring of the vaginal discharge. The body condition score and milk production were recorded on the day of sampling. The biochemical profile encompassed albumin, urea, gamma‐glutamyl transferase, calcium, fibrinogen and cholesterol concentrations. The number of services per pregnancy was lower (p < 0.01), and the number of days until first insemination and the median time to pregnancy were higher in cows with PVD (p < 0.01) when compared with control cows. Milk production and body condition score were lower (p < 0.01) in cows with PVD than in control group. Cows with PVD had lower (p < 0.05) serum albumin, urea, calcium and cholesterol concentrations, and higher serum gamma‐glutamyl transferase activity and fibrinogen concentration than cows without PVD. Our results show that cows with PVD have changes in the biochemical profile and negative effects on production and reproduction performance.     

Ovarian response to prostaglandin F2α in lactating dairy cows: A clinical update

Prostaglandin F_2α (PGF_2α) and its analogs are used to induce luteolysis in estrus synchronization programs to terminate unwanted pregnancies or to promote ovulation in certain cow subpopulations. In the past few decades, the luteolytic dose of PGF_2α has remained unchanged. This review explores the clinical implications of increasing the standard dose for these applications in high-producing dairy cows. Ultrasonography may assist in selecting the most appropriate PGF_2α dose and improve the results. A reference has been used for PGF_2α for promoting ovulation in herds showing poor reproductive performance.     

Morphological Quality of Oocytes and Blood Plasma Metabolites in Repeat Breeding and Early Lactation Dairy Cows

The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea.     

Clinical prospects proposing an increase in the luteolytic dose of prostaglandin F2α in dairy cattle

Over the past few decades, the luteolytic dose of prostaglandin F_2α (PGF_2α) and its analogs, used to synchronize estrus for fixed-time insemination in dairy cattle, have remained unchanged. Given the beneficial effects of PGF_2α on a young corpus luteum and on multiple ovulations in a fixed-time insemination protocol, and its therapeutic abortive effects on multiple ovulations in pregnant cows, we propose the use of a double PGF_2α dose or two PGF_2α treatments 24 hours apart. Ultrasonography procedures serve to identify luteal structures and may therefore help to determine the best PGF_2α dose to improve the fertility of high-producing dairy cows.     

Fecal and urinary lignans, intrafollicular estradiol, and endometrial receptors in lactating dairy cows fed diets supplemented with hydrogenated animal fat, flaxseed or sunflower seed

We hypothesized that the inclusion of flaxseed in the diets of lactating dairy cows will increase urinary and fecal concentrations of the lignans, secoisolariciresinol diglycoside (SDG), enterolactone and enterodiol, reduce intrafollicular concentrations of IGF-I and estradiol, and subsequently reduce estradiol and oxytocin receptor expression in the endometrium. To test this hypothesis, 27 cycling, lactating Holstein cows were assigned to 1 of 3 diets supplemented with saturated fatty acids (SAT), flax (FLX), or sunflower (SUN) seed. Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized, and 5 d later, follicles>8 mm were ablated by an ultrasound-guided procedure in all cows. Samples of blood (Days 0 to 14), follicular fluid (Day 5 and 15), endometrium (Day 15), as well as urine and feces were collected in a subset of the animals. The fecal concentrations of SDG and enterodiol were higher (P<0.05) in cows fed FLX than in those fed SAT or SUN. Enterodiol increased (P<0.05) in urine samples of cows fed FLX, compared to those of cows fed SUN. However, follicular estradiol concentrations on Day 5 and 15 and endometrial concentrations of estradiol and oxytocin receptors on Day 15 did not differ among the dietary groups. Mean plasma progesterone concentrations were higher (P<0.05) in cows fed FLX and SUN than in those fed SAT. In summary, a diet supplemented with flaxseed increased the concentrations of SDG and enterodiol in feces, as hypothesized, but did not alter intrafollicular concentrations of IGF-I or estradiol, or endometrial populations of oxytocin or estrogen receptors in lactating dairy cows.     

Association of endometritis and ovarian follicular cyst with mastitis in dairy cows

The occurrence of multiple metabolic and inflammatory diseases in dairy cows is higher during the periparturient period, which may be triggered by bacterial components, but not a viable bacterium. This study aimed to determine the association of endometritis and ovarian follicular cyst (OFC) with mastitis in dairy cows. Ninety-eight Holstein dairy cows were clinically examined for endometritis and OFC approximately 30–50 days after calving. Blood and milk samples were collected for the determination of milk somatic cell count (SCC); milk interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and interleukin-8 (IL-8) concentrations; and plasma haptoglobin (Hp) and lipopolysaccharide-binding protein (LBP) concentrations. Of the 98 dairy cows included in this study, 12 were diagnosed with endometritis and 37 cows were identified as OFC-positive, whereas the remaining 49 cows were healthy (without endometritis or OFC). The average and maximum SCCs and plasma Hp and LBP concentrations were not significantly different between the healthy cows and those with endometritis or OFC. However, when the maximum SCC was classified as <300, 300–1,000, or >1,000 × 10³ cells/ml, the percentage of cows with the maximum SCC <300 × 10³ cells/ml was significantly lower in the endometritis and OFC-positive groups than in the healthy group. These results suggested that cows with endometritis and OFC during the postpartum period exhibit high SCC, indicating that some bacterial components can be transferred between organs.     

Metabolic parameters and their relationship to energy balance in multiparous Simmental,Brown Swiss and Holstein cows in the periparturient period as influenced by energy supply pre‐ and post‐calving

A study was conducted to evaluate the effects of three energy supply (E) levels [low (L), medium (M), high (H)], both pre‐partum (PRE) and post‐partum (POST), and their interactions on metabolic parameters and energy balance (EB) in dairy cows of three breeds. In both phases, E levels applied to a total of 81 multiparous cows of breeds Simmental (SI), Brown Swiss (BS) and Holstein–Friesian (HF; n = 27 for each breed) were 75%, 100% and 125% of recommendations of the German Society of Nutrition Physiology, using a 3 × 3 factorial arrangement of treatments. During the pre‐calving period, serum concentrations of non‐esterified fatty acids (NEFA) were higher for L_PRE cows, and glucose concentrations were elevated for H_PRE cows. During the lactation period, NEFA concentrations were greatest for treatment L_POST. Mean concentrations of β‐hydroxybutyrate (BHB) were highest for cows of the L_POST treatment, intermediate for M_POST and lowest for H_POST. Glucose concentrations were lower for L_POST cows. SI cows had lower BHB concentrations both pre‐ and post‐calving and higher glucose concentrations during early lactation than the other breeds. BHB concentration POST was highest for BS cows. Restricted feeding PRE resulted in a better energy status of cows fed above energy requirements POST (E_PRE × E_POST interaction). HF cows had a higher EB pre‐calving, whereas SI cows had a less negative EB during early lactation, compared with the other breeds respectively. Correlations of serum NEFA, BHB and glucose concentrations with EB were strongest during the transition period. Results suggest that controlling energy intake during the dry period might be advantageous for the energy status of dairy cows after calving, whereas energy restriction in early lactation leads to metabolic stress. Evidence is provided of a clear relationship between EB and the blood metabolites NEFA and BHB, especially in the transition period.     

高产奶牛繁殖障碍的起因和调控因素

奶牛的管理、营养、产量和基因选择都与其生育能力有直接关系.从体况评分、营养信号、内分泌和生殖器官等方面综述了高产奶牛繁殖障碍的起因和调控因素.     

Progesterone Profiles in the Caudal Vena Cava and Jugular Vein in Response to Pulsatile Luteinizing Hormone Stimulation Induced by GnRH Treatment During the Mid-luteal Phase in Lactating Dairy Cows

The aim of this study was to examine whether increased frequency of luteinizing hormone (LH) pulses influences luteal progesterone (P₄) secretion by measuring progesterone concentrations at the secreted (caudal vena cava) and circulating levels (jugular vein) in lactating dairy cows. Cows received six intravenous administrations of 2.5 μg of GnRH (gonadorelin acetate, n=4) or 2 ml saline (n=3) at 1-h intervals on 12.4 ± 0.4 (mean ± SE) days after ovulation. Blood samples were collected from the caudal vena cava and jugular vein every 12 min for 12 h (6 h before and after treatment). During the 6 h after treatment, frequency of LH pulses (5.3 ± 0.3 and 3.0 ± 0.0 pulses/6 h) and mean LH concentration (0.50 ± 0.06 and 0.38 ± 0.05 ng/ml) were greater (P<0.05) in GnRH-treated cows than in saline-treated cows. Mean P₄ concentration and amplitude of P₄ pulses in the caudal vena cava during the 6 h after treatment were greater (P<0.05) in GnRH-treated cows than in saline-treated cows, but the frequency of P₄ pulses was not different between the groups. Mean P₄ concentration in the jugular vein during the 6 h after treatment was also higher (P<0.05) in GnRH-treated cows than in saline-treated cows (7.0 ± 1.3 and 5.4 ± 0.9 ng/ml). These results indicate that the increased frequency of LH pulses stimulates progesterone secretion from the functional corpus luteum and brings about higher P₄ concentrations in the circulating blood in lactating dairy cows.     

Comparison of two types of CIDR-based timed artificial insemination protocols for repeat breeder dairy cows

This study compared two types of controlled internal drug release (CIDR)-based timed artificial insemination (TAI) protocol for treatment of repeat breeder dairy cows. In the first trial of the experiment, 55 repeat breeder cows were randomly assigned to the following two treatments. (1) In the EB group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 1 mg estradiol benzoate (EB) plus 50 mg progesterone (P4; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 1 mg EB on Day 8 and were subjected to TAI 30 h later (n=27). (2) In the gonadotrophin releasing hormone (GnRH) group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 250 microg gonadorelin (GnRH; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 250 microg GnRH on Day 9 and were subjected to TAI 17 h later (n=28). In the second trial, 41 repeat breeder cows that were confirmed as not pregnant in the first trial were randomly assigned to the same two treatments used in the first trial (an EB group of 20 cows and a GnRH group of 21 cows). The ovaries of 15 cows from each group were examined by transrectal ultrasonography in order to observe the changes in ovarian structures, and blood samples were collected for analysis of serum P4 concentrations. The pregnancy rates following TAI in the first (18.5 vs. 32.1%) and second (40.0 vs. 38.1%) trials and the combined rates (27.7 vs. 34.7%) did not differ between the EB and GnRH groups. The proportions of cows with follicular wave emergence within 7 days did not differ between the EB (12/15) and GnRH groups (13/15). The interval to wave emergence was shorter (P<0.01) in the GnRH group than in the EB group, but there was no difference in the mean diameters of dominant follicles on Day 7 between the groups. Moreover, the proportions of cows with synchronized ovulation following a second EB or GnRH treatment did not differ between the groups. In conclusion, treatment with either EB or GnRH in a CIDR-based TAI protocol results in synchronous follicular wave emergence, follicular development, synchronous ovulation, and similar pregnancy rates for TAI in repeat breeder cows.     

Comparison of cholesterol levels among lipoprotein fractions separated by anion-exchange high-performance liquid chromatography in periparturient Holstein–Friesian dairy cows

Changes in lipoprotein profiles occur in dairy cows during the periparturient period and in cows with transition cow disease. Here, the lipoprotein profiles of Holstein–Friesian dairy cows during the periparturient period were obtained by anion-exchange, high-performance liquid chromatography to evaluate the usefulness of lipoprotein profile evaluation during the periparturient period and in cows with fatty liver and milk fever. Lipoprotein levels (including total and high- (HDL-C) and low-density lipoprotein (LDL-C) cholesterol) in 10 healthy cows were low 4 weeks prepartum, with the lowest values at calving or within 1 week of calving; the values increased at 8 weeks postpartum. The lipoprotein levels were measured in 16 cows diagnosed with fatty liver (n=10) or milk fever (n=6) and compared to 10 healthy dairy cows. A significant difference was observed in HDL-C between healthy cows (at calving and 1 week postpartum), and the fatty liver and milk fever cows. Cows with fatty liver and milk fever had a lower mean HDL-C than the 10 healthy dairy cows at calving and 1 week postpartum. HDL-C might be a good indicator of energy balance for differentiating healthy cows from those with transition cow disease.