BEHAVIORAL, PHYSIOLOGICAL AND ADRENAL RESPONSES TO REGROUPING AND RELOCATION IN SHEEP

Document Type : Research article

Authors

Department of Animal hygiene, Faculty of Veterinary Medicine, Assiut University

Abstract

Forty non-pregnant and non-lactating multiparus ewes of the local Ossemi breed, about 4 years in age and 50 kg in weight were used in this investigation. They were divided into four groups each of 10 ewes.
Assiut Vet. Med. J. Vol. 52 No. 109 April 2006
Animals in each group were housed together under normal environmental conditions in a separate well-ventilated and well-lighted straw-bedded pen. All groups were subjected to a 10 days preliminary period for group acclimatization, stability and welfare. After that, ewes in the first group were used as control animals where they were never moved from their pen and no more animals were added. However, animals of the second group were used for making regrouping with that of the third group or regrouping and relocation with that of the fourth one for three times during the studying period at weekly intervals. Ewes were ad libitum fed on commercial concentrate mixture and wheat straw and average daily food intake was calculated, however drinking water was freely available allover the experiment. Behavioral pattern of the experimented animals was recorded at each social and pen exchange. Moreover, they were clinically examined to determine their average pulse rate, respiratory rate and body temperature. Blood samples were collected to measure their serum cortisol level. The obtained results indicated that, moving and merging of sheep through regrouping and regrouping with relocation were resulted in aversive environmental conditions and leaded to increased aggression and consequent injuries and reflected prominently on their behavior, food intake and serum cortisol level. Therefore, stability of the herd as well as avoidance of moving and merging as much as possible should be considered during
establishing their farms.

Keywords


Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Department of Animal hygiene, Faculty of Veterinary Medicine, Assiut University

BEHAVIORAL, PHYSIOLOGICAL AND ADRENAL

RESPONSES TO REGROUPING AND RELOCATION IN SHEEP

(With 4 Tables and 3 Figures)

By M.A. ABDEL-RAHMAN and MADEHA H.A. DARWISH

(Received at 5/3/2006)

الاستجابات السلوکية والفسيولوجية والهرمونية والناتجة عن إعادة تجميع

وتسکين الأغنام معتز أحمد محمد عبد الرحمن،مديحه حسنی أحمد درویش تم إجراء هذه التجربة على عدد 40 من النعاج الغير حوامل أو مرضع من سلالة أغنام محلية الأوسيمي) والمتماثلة في العمر والوزن تقریبا وسبق لها الولادة عدة مرات. قسمت هذه الحيوانات إلى أربعة مجموعات متساوية وتم تسکين کل مجموعة على حدة في حظائر منفصلة تحت الظروف البيئية السائدة لمدة10 أيام

کفترة تمهيدية. أستخدمت حيوانات المجموعة الأولى کمجموعة ضابطة حيث لم يتم إضافة أو نقل حيوانات من أو إلى هذه المجموعة بينما أستخدمت حيوانات المجموعة الثانية لاختبار إعادة التجميع مع المجموعة الثالثة أو إعادة التجميع والتسکين مع المجموعة الرابعة. تم تغذية هذه الحيوانات طوال فترة التجربة على قش القمح و مخلوط المرکزات مع توافر ماء الشرب بحرية وتم تحديد متوسط الأکل اليومي لها. تم تسجيل السلوکيات المختلفة لهذه النعاج وذلک مع کل إعادة تجميع أو تسکين. بالإضافة إلى هذا، تم فحص الحالة الصحية للحيوانات المستخدمة في هذه التجربة وذلک لتعيين معدلات النبض والتنفس ودرجة حرارة جسم هذه الحيوانات. کذلک أخذت عينات من دم هذه الحيوانات في بداية کل مرحلة تجريبية وعوملت هذه العينات المعاملة الخاصة لتعيين نسبة هرمون الکورتيزول بها. أثبتت النتائج التي تم الحصول عليها أن إعادة التجميع أو إعادة التجميع والتسکين بالنسبة للأغنام کانت من العوامل الضاغطة والتي أتت بانعکاسات سلبية مباشره على السلوکيات المختلفة ومتوسط الأکل اليومي لها وکذلک نسبة هرمون الکورتيزول بدم هذه الحيوانات. وقد نوهت هذه الدراسة عن مدى أهمية تجنب إعادة تجميع الأغنام داخل نفس الحظائر أو إعادة التجميع والتسکين داخل حظائر جديدة وذلک للوصول للمستوى الإنتاجي المطلوب.

SUMMARY

Forty non-pregnant and non-lactating multiparus ewes of the local Ossemi breed, about 4 years in age and 50 kg in weight were used in this investigation. They were divided into four groups each of 10 ewes.

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Animals in each group were housed together under normal environmental conditions in a separate well-ventilated and well-lighted straw-bedded pen. All groups were subjected to a 10 days preliminary period for group acclimatization, stability and welfare. After that, ewes in the first group were used as control animals where they were never moved from their pen and no more animals were added. However, animals of the second group were used for making regrouping with that of the third group or regrouping and relocation with that of the fourth one for three times during the studying period at weekly intervals. Ewes were ad libitum fed on commercial concentrate mixture and wheat straw and average daily food intake was calculated, however drinking water was freely available allover the experiment. Behavioral pattern of the experimented animals was recorded at each social and pen exchange. Moreover, they were clinically examined to determine their average pulse rate, respiratory rate and body temperature. Blood samples were collected to measure their serum cortisol level. The obtained results indicated that, moving and merging of sheep through regrouping and regrouping with relocation were resulted in aversive environmental conditions and leaded to increased aggression and consequent injuries and reflected prominently on their behavior, food intake and serum cortisol level. Therefore, stability of the herd as well as avoidance of moving and merging as much as possible should be considered during

establishing their farms.

Key words: Behavior, physiology, adrenal, regrouping, relocation, sheep

INTRODUCTION Many conventional management practices may be stressful for livestock. This stressful situations annoy the animals and a brief period of it may cause a variety of physiological changes that result in poor production performance and reduced defense mechanisms against diseases. So, it is considered as a managemental stresses (Klastrup et al., 1987 and Fraser & Broom, 1990).

Stress is defined as an external body forces that tend to disp.ace the homeostatic state of the animal (Scott, 1981). In lactating animals, stressful events can hamper milk secretion and ejection reflex and may enhance the risk of mastitis (Bruckmaier et al., 1993). The inhibition of the milk ejection reflex is a consequence of activation of the sympathetic-adrenal system, which reduces the release of oxytocin and its access to the mammary epithelium (Findlay and Grosvenor, 1969; Goodman and Grosvenor, 1983 & Bruckmaier and Blum, 1992).

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Moreover, increased level of plasma corticosteriods in stressed animals may impair the immune functions and reduce the synthesis of milk proteins due to amino acid utilization for gluconeogenesis (Hart, 1983; Napolitano et al., 1995 and Rhind et al., 1998).

The disruption of suitable social links may lead to increased aggression and consequent injuries as well as reduced growth rates (Friend et al., 1983 and Rushen, 1987). Moving and merging were found to cause increased cortisol secretion and to have slight and short term effect on the productive traits (Varner and Johnson, 1983 and Hasegawa et al., 1997).

Indeed, sheep are gregarious and relatively defenseless animals and any husbandry operations may result in the motivational state of fear, anxiety and frustration (Lynch et al., 1992 and Casamassima et al., 2001). So, the present study focused on the stressfulness of regrouping and regrouping with relocation and its effect on behavioral, physiological and adrenal responses in sheep.

MATERIALS and METHODS

I- Animals, experimental design and management: -

Forty non-pregnant and non-lactating multiparus ewes of the local Ossemi breed, nearly of about 4 years in age and 50 kg in weight were used in this investigation. They were divided into four groups each of 10 ewes. Animals in each group were housed together under normal environmental conditions in a separate well-ventilated and well-lighted straw-bedded pen. Each pen was provided with a manger and water trough with a sufficient feeding and watering space per animal. Used pens were selected to be faraway from each other so that, animals in each pen were isolated from tactile, visual and auditory stimuli of animals in other pens.

The experiment was designed according to Sevi et al. (2001). All groups were subjected to a 10 days preliminary period for group acclimatization, stability and welfare. After that, ewes in the first group were used as control animals where they were never moved from their pen and no more animals were added. However, animals of the second group were used for making regrouping with that of the third group or regrouping and relocation with that of the fourth one for three times during the studying period at weekly intervals (day 1, 8, 15). Animals of the third and fourth groups were individually marked for further behavioral observations and experimental analyses. At early morning of these initiative days, five ewes of the second group replaced others of

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

the third group which never moved from their pens (regrouping) while other animals of the second group replaced others of the fourth group and all members were moved to a new pen of the same conditions (regrouping and relocation).

Allover the experiment, ewes were ad libitum fed on commercial concentrate mixture and wheat straw and feed refusal was weighed daily before next morning feeding and was deducted from the total offered weight to calculate the daily intake. Water was freely available allover the experiment. II- Behavioral observations and measurements: -

Ewes were fed and cleaned out at 8:00 a.m. and during the remainder of the day humans were not normally present. Their behavioral pattern was recorded according to Marten and Bateson (1988); Fordham et al. (1991) and Sevi et al. (2001) using the scan sampling technique in which a screen was erected at one end of the room so that the observer can study all the animals at the same time without being seen by them. Three behavioral recordings were conducted after each social and pen exchange (day 1, 8 and 15). Experimented ewes were observed for nine hours per day (three hours per group). Each group was observed for one hour in the morning (9:00 to 12); one hour in the afternoon (13:00 to 16:00) and one hour around dusk (17:00 to 20:00). Animals were observed for their postural situation standing or lying); behavioral activity (walking, eating, ruminating, drinking or not active) as well as social and aggressive interactions (butting, chasing, sniffing each other). Postural situation and behavioral activities were expressed as percentage of the observation / recorded hour. Meanwhile, social and aggressive interactions are short lasting events so, they were expressed as number of total observations / day. III- Physiological measurements: -

At the initiative day of each social and pen exchange and just before starting blood sampling and behavioral recording, experimented ewes were clinically examined according to Blood and Radostits (1990) to determine their average pulse rate, respiratory rate and body temperature.

IV- Blood sampling: -

At each social and pen exchange, Blood samples were collected from the jugular vein of the experimented animals at the initiative day just before starting the behavioral recording. Samples were allowed to coagulate at room temperature and serum separated by centrifugation.

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

The sera were freezed at -20 °C till further analysis to determine its cortisol level using TDXFLX system with fluorescence polarization and competitive binding technique according to Dandliker & Feigen (1970) and Dandliker & Saussure (1973).

V- Statistical analysis: -

Statistical analyses of the collected data were carried out according to procedures of completely random design, SAS (1995).

RESULTS

Item

1000

The results of this study were illustrated in tables 1,2,3,4 as well as Figures 1, 2 and 3. Table 1: Postural situation and behavioral activities of the experimented

ewes

Treatment Control Regrouping Regrouping and relocation

------ Postural situation /hour) -------- Standing

602

705 Lying

00 ---- Behavioral activities % /hour) -- Walking Eating Ruminating

209 Drinking

10 No activities

10 Aggressive interactions

18+2

21+2 (No./day)

Figures in the same raw with different superscripts differs significantly (p<0.01)

402

306

306

30b

30*

10

% /hour

Sitting

Walking

Eating

Drinking

No activities

Ruminating

Aggr. inter. (No./day)

Control

Regrouping Regrouping and relocation Fig. 1: Postural situation and behavioral activities of the experimented

ewes.

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Table 2: Changes in voluntary food intake of the experimented ewes.

Treatment

Day

Control

Regrouping

Regrouping and

relocation

---------- Average (g/day) ------

1730+25

· 1250+50

930+25

1710+50

1260230

980:20

1690+45

1290+10

1050+20

1700+20

1330230

1130435

1720-25

1370+50

1180250

1680+35

1250–35

1510+20

1550+20

1700+20

127050

Average

1704+31a

1366+306

1113+34°

Figures in the same raw with different superscripts differs significantly (p<0.01).

2000

1800

1600

1400

g/day

1st

2nd

3rd

5th

6th

7th

4th days

Control

---Regrouping

Regrouping and relocation

Fig. 2: Changes in voulantry food intake of the experimented ewes.

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Table 3: Changes in the physiological status of the experimented ewes

Treatment

Items

Control

Regrouping

Regrouping and

relocation

74+4

82+2

84+4

Pulse rate

(No./min.) Respiratory rate

(No./min.) Body temperature

18+

22+2

26+2

39.1+0.1

39.3+0.1

39.180.1

(°C)

Table 4: Changes in serum cortisol level of the experimented ewes

Treatment Item

Control

Regrouping and Regrouping

relocation ---------- Average (ug/100 ml) ------ Cortisol level

0.47+0.01

0.71+0.01

0.94+0.01

Figures in the same raw with different superscripts differs significantly (p <0.01)

Ug/100ml

!

WH

W

SASULULU

Control

Regrouping

Regrouping and relocation

Fig. 3: Changes in serum cortisol level of the experimented ewes

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

DISCUSSION

I- Behavioral observations of experimented animals: -

The data represented in table (1) and assimilated on figure (1) showed the effect of the studied regrouping as well as regrouping and relocation on the behavior of the experimented ewes. These data revealed that, both of postural situation, behavioral activities and social interactions were significantly affected with the studied conditions (P<0.01).

The percentage of ewes that were recorded in different postural situation was 60; 70 and 100 in standing and 40, 30 & 0 in lying or idling following control; regrouping and regrouping with relocation, receptively. Locomotiom activity was higher for sheep subjected to group and pen exchange than for those subjected to regrouping or control conditions. The percentage of ewes that were recorded walking during the observation time were 0, 30 and 80 following control; regrouping and regrouping with relocation, respectively.

Eating and rumination activities were also affected with these treatments. The percentage of ewes that were recorded eating or ruminating was 50, 30, 10 and 30, 20, 0 following control; regrouping and regrouping with relocation, receptively. However, the percentage of ewes that were recorded drinking or not active during the observation time was not significantly affected following these treatments and the values were 10, 10,10 and 10, 10, 0 following control; regrouping and regrouping with relocation, receptively.

The number of aggressive interactions during the recorded time was significantly higher after member exchange following regrouping and regrouping with relocation. The data were 4, 18 and 21 No. / day for control, regrouping and regrouping with relocation, respectively.

It has been stated that, high levels of locomotor activities may reflect the presence of aversive environmental conditions (Fraser and Broom, 1990 and Barnett et al., 1992). Moreover, Grasso et al. (1999) observed a higher incidence of resting behaviors, as lying and idling, in less stressful environments. The results of the present study agreed with these findings. The percentage of ewes spent the time in standing was significantly higher after regrouping with relocation and followed by those subjected to regrouping and control conditions, respectively. Animals subjected to regrouping spent most of its standing time in eating and walking, however, regrouped and relocated ewes spent most of their standing time in walking. This finding may be related to the new

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

aversive environmental conditions of regrouping and relocation where the animals may induced by forced social interactions to fight or escape and active behaviors may be adopted to get readily prepared to react (Hanlon et al., 1994 and Sevi et al., 2001).

In the same time, the obtained results indicated that, aggressive behavioral interactions were significantly increased either with regrouping or regrouping and relocation. This finding agreed with Tennessen et al. (1985), Kondo and Hurnic (1990) and Sevi et al. (2001) who indicated that, aggression can be employed to gain more food or a higher-quality food or to establish a place in a social hierarchy with new situations and it lasts about 4 days after strange animals are mixed. II- Food intake of the experimented animals: -

The data represented in table (2) showed the effect of the studied regrouping as well as regrouping and relocation on voluntary food intake of the experimented ewes. These data revealed that, average food intake of the experimented ewes was significantly affected with the studied conditions (P<0.01). Average food intake of the experimented ewes was 1704; 1366 and 1113 g/day for control; regrouping and regrouping with relocation, respectively. Moreover, Fig. (2) showed that, daily food intake of the experimented ewes was the lowest allover the experiment for those subjected to regrouping with relocation while it was markedly lower for those subjected to regrouping only as compared to control ones. However, the effect of regrouping and regrouping with relocation on food intake was gradually decreased from the 1st to the 7th day of the experiments. This finding agreed with Varner et al. (1983); Hanlon et al. (1994) and Sevi et al. (2001) and could be attributed to higher aggression and unfamiliarity to the new aversive environmental conditions of regrouping and regrouping with relocation. III- Physiological status of the experimented animals: -

The data represented in table (3) showed the average pulse rate, respiratory rate (No. 1 min.) and body temperature (°C) of the experimented ewes. It was 74, 18, 39.1; 82, 22, 39.3 and 84, 26, 39.1 after control, regrouping and regrouping with relocation, respectively. These results indicated that, non-of the studied conditions had a significant effect on physiological and health status of the experimented ewes. However, the slight increase in their pulse and respiratory rates after regrouping and regrouping with relocation was within the normal range and could be attributed to the physiological and biological adjustments and changes in the animal body to meet that new stressful situation (Hafez, 1975; Banerjee, 1982 and Radostits et al., 1994).

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Hafez, E.S. (1975): The behavior of domestic animals. 3rd Ed.,

Bailliere-Tindall-London. Hanlon, A.J.; Rhind, S.M.; Reid, H.W. and McMillen, S.R. (1994):

Relationship between immune response, live weight gain. behavior and adrenal function in red deer calves derived from

wild and farmed stock. Appl. Anim. Behav. Sci., 41: 243-255. Hart, I.C. (1983): Endocrine control of nutrient partition in lactating

ruminants. Proc. Nutr. Soc., 42: 181-194. Hasegawa, N.; Nishiwaki, A.; Sugawara, K. and Ito, I. (1997): The

effect of social exchange between two groups of lactating primiparous heifers on milk production, dominance order, behavior and adrenocortical response. Appl. Anim. Behav. Sci.,

51: 15-27. Hopster, H.; Van der Werf, J.; Erkens, J. and Blohkius, H. (1999):

Effects of repeated jugular puncture on plasma cortisol concentrations in loose-housed dairy cows. J. Anim. Sci., 77:

708-714. Klastrup, O.J.; Bakken, J.B. and Bushnell, R. (1987): Environmental

influences on bovine mastitis. FIL-IDF Bulletin no. 217,

Brussels, Belgium. Kondo, B. and Hurnik, J.F. (1990): Stabilization of social hierarchy in

dairy cows. Appl. Anim. Behav. Sci., 27:287-297. Lynch, J.J.; Hich, G.N. and Adms, D.B. (1992): The behavior of sheep.

Biological principles and implications for production. CAB

International, Wallingford, UK. Marten, P. and Bateson, P. (1988): In Measuring behaviour. Cambridge

University Press, Cambridge, pp. 48-69. McDonalds, L.E. (1969): Veterinary endocrinology and reproduction.

1st Ed., Lea and Febiger, Philadelphia, USA. Napolitano, F.; Marino, V.; De Rosa, G. and Bordi, A. (1995):

Influence of artificial rearing on behavioural and immune

response of lambs. Appl. Anim. Beav. Sci., 45: 245-253. Radostits, O.M.; Leslie, K.E. and Fetrow, J. (1994): Herd health. 2dn

Ed., UK Rhind, S.M.; Reid, H.W.; Mc Millen, S.R. and Palmarini, G. (1998): The

role of cortisol and B-endorphin in the response of the immune

system to weaning in lambs. Anim. Sci., 66: 397-402. Rushen, J. (1987): A difference in weight reduces fighting when

unacquainted newly weaned pigs first meet. Can. J. Anim. Sci., 67: 951-960.

B1010

12

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

SAS (1995): Statistical analysis system. User's Guide : Statistics.

Version 6, 2nd Ed., SAS Inst. Inc., Cary, NC. Scott, G.H. (1981): What is animal stress and how is measured ?, J.

Anim. Sci., 52: 150-153. Sevi, A., Taibi, L.; Albenzio M.; Muscio A., Dell S and Napolitano F.

(2001): Behavioral, adrenal, immune and productive responses of lactating ewes to regrouping and relocation. J. Anim. Sci.,

79: 1457-1465. Tennessen, T.; Price, M.A. and Berg, RT. (1985): The social interaction

of young bulls and stress after re-grouping. Appl. Anim. Behav.

Sci., 14:37-47. Varner, M.A. and Johnson, B.H. (1983): Influence of

adrenocotricotropin upon milk production, milk constituents and endocrine measures of dairy cows. J. Dairy Sci., 66: 458

465. Varner, M.A.; Johnson, B.H. and Mochrie, R.D. (1983): Influence of

herd relocation upon production and endocrine traits of dairy cows. J. Dairy Sci., 66: 466-474.

13

Hafez, E.S. (1975): The behavior of domestic animals. 3rd Ed.,
Bailliere-Tindall-London. Hanlon, A.J.; Rhind, S.M.; Reid, H.W. and McMillen, S.R. (1994):
Relationship between immune response, live weight gain. behavior and adrenal function in red deer calves derived from
wild and farmed stock. Appl. Anim. Behav. Sci., 41: 243-255. Hart, I.C. (1983): Endocrine control of nutrient partition in lactating
ruminants. Proc. Nutr. Soc., 42: 181-194. Hasegawa, N.; Nishiwaki, A.; Sugawara, K. and Ito, I. (1997): The
effect of social exchange between two groups of lactating primiparous heifers on milk production, dominance order, behavior and adrenocortical response. Appl. Anim. Behav. Sci.,
51: 15-27. Hopster, H.; Van der Werf, J.; Erkens, J. and Blohkius, H. (1999):
Effects of repeated jugular puncture on plasma cortisol concentrations in loose-housed dairy cows. J. Anim. Sci., 77:
708-714. Klastrup, O.J.; Bakken, J.B. and Bushnell, R. (1987): Environmental
influences on bovine mastitis. FIL-IDF Bulletin no. 217,
Brussels, Belgium. Kondo, B. and Hurnik, J.F. (1990): Stabilization of social hierarchy in
dairy cows. Appl. Anim. Behav. Sci., 27:287-297. Lynch, J.J.; Hich, G.N. and Adms, D.B. (1992): The behavior of sheep.
Biological principles and implications for production. CAB
International, Wallingford, UK. Marten, P. and Bateson, P. (1988): In Measuring behaviour. Cambridge
University Press, Cambridge, pp. 48-69. McDonalds, L.E. (1969): Veterinary endocrinology and reproduction.
1st Ed., Lea and Febiger, Philadelphia, USA. Napolitano, F.; Marino, V.; De Rosa, G. and Bordi, A. (1995):
Influence of artificial rearing on behavioural and immune
response of lambs. Appl. Anim. Beav. Sci., 45: 245-253. Radostits, O.M.; Leslie, K.E. and Fetrow, J. (1994): Herd health. 2dn
Ed., UK Rhind, S.M.; Reid, H.W.; Mc Millen, S.R. and Palmarini, G. (1998): The
role of cortisol and B-endorphin in the response of the immune
system to weaning in lambs. Anim. Sci., 66: 397-402. Rushen, J. (1987): A difference in weight reduces fighting when
unacquainted newly weaned pigs first meet. Can. J. Anim. Sci., 67: 951-960.
B1010
12
Assiut Vet. Med. J. Vol. 52 No. 109 April 2006
SAS (1995): Statistical analysis system. User's Guide : Statistics.
Version 6, 2nd Ed., SAS Inst. Inc., Cary, NC. Scott, G.H. (1981): What is animal stress and how is measured ?, J.
Anim. Sci., 52: 150-153. Sevi, A., Taibi, L.; Albenzio M.; Muscio A., Dell S and Napolitano F.
(2001): Behavioral, adrenal, immune and productive responses of lactating ewes to regrouping and relocation. J. Anim. Sci.,
79: 1457-1465. Tennessen, T.; Price, M.A. and Berg, RT. (1985): The social interaction
of young bulls and stress after re-grouping. Appl. Anim. Behav.
Sci., 14:37-47. Varner, M.A. and Johnson, B.H. (1983): Influence of
adrenocotricotropin upon milk production, milk constituents and endocrine measures of dairy cows. J. Dairy Sci., 66: 458
465. Varner, M.A.; Johnson, B.H. and Mochrie, R.D. (1983): Influence of
herd relocation upon production and endocrine traits of dairy cows. J. Dairy Sci., 66: 466-474.