Group Title: BMC Veterinary Research
Title: Pregnancy success of lactating Holstein cows after a single administration of a sustained-release formulation of recombinant bovine somatotropin
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Title: Pregnancy success of lactating Holstein cows after a single administration of a sustained-release formulation of recombinant bovine somatotropin
Physical Description: Book
Language: English
Creator: Bell, A.
Rodríguez,O. A.
de Castro e Paula, L. A.
Padua, M. B.
Hernández-Cerón, J.
Gutiérrez, C. G.
De Vries, A.
Hansen, P. J.
Publisher: BMC Veterinary Research
Publication Date: 2008
 Notes
Abstract: BACKGROUND:Results regarding the use of bovine somatotropin for enhancing fertility in dairy cattle are variable. Here, the hypothesis was tested that a single injection of a sustained-release preparation of bovine somatotropin (bST) during the preovulatory period would improve pregnancy success of lactating dairy cows at first service.RESULTS:The first experiment was conducted in a temperate region of Mexico. Cows inseminated following natural estrus or timed artificial insemination were given a single injection of bST or a placebo injection at insemination (n = 100 cows per group). There was no significant difference between bST and control groups in the proportion of inseminated cows diagnosed pregnant (29 vs 31% pregnant). The second experiment was performed during heat stress in Florida. Cows were subjected to an ovulation synchronization regimen for first insemination. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. As expected, bST did not increase vaginal temperature. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124–132 cows per group). There was a tendency (p = 0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (= 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control).CONCLUSION:Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows.
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Research article


Pregnancy success of lactating Holstein cows after a single
administration of a sustained-release formulation of recombinant
bovine somatotropin
A Bell', OA Rodriguez2, LA de Castro e Paula', MB Padua', J Hernandez-
Cer6n2, CG Gutierrez2, A De Vries' and PJ Hansen*


Address: 'Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910, USA and 2Departamento de Reproducci6n,
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut6noma de Mexico, Ciudad Universitaria, DF, CP 04510, Mexico
Email: A Bell adrianeb@ufl.edu; OA Rodriguez mvzarmandorc@yahoo.com.mx; LA de Castro e Paula luizacp@ufl.edu;
MB Padua mpadua@ufl.edu; J Hernandez-Cer6n jhc@servidor.unam.mx; CG Gutierrez ggcarlos@servidor.unam.mx; A De
Vries devries@ufl.edu; PJ Hansen* hansen@animal.ufl.edu
* Corresponding author



Published: 26 June 2008 Received: 9 January 2008
BMC Veterinary Research 2008, 4:22 doi: 10.1 186/1746-6148-4-22 Accepted: 26 June 2008
This article is available from: http://www.biomedcentral.com/1746-6148/4/22
2008 Bell et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Results regarding the use of bovine somatotropin for enhancing fertility in dairy
cattle are variable. Here, the hypothesis was tested that a single injection of a sustained-release
preparation of bovine somatotropin (bST) during the preovulatory period would improve
pregnancy success of lactating dairy cows at first service.
Results: The first experiment was conducted in a temperate region of Mexico. Cows inseminated
following natural estrus or timed artificial insemination were given a single injection of bST or a
placebo injection at insemination (n = 100 cows per group). There was no significant difference
between bST and control groups in the proportion of inseminated cows diagnosed pregnant (29 vs
31% pregnant). The second experiment was performed during heat stress in Florida. Cows were
subjected to an ovulation synchronization regimen for first insemination. Cows treated with bST
received a single injection at 3 days before insemination. Controls received no additional treatment.
As expected, bST did not increase vaginal temperature. Treatment with bST did not significantly
increase the proportion of inseminated cows diagnosed pregnant although it was numerically
greater for the bST group (24.2% vs 17.8%, 124-132 cows per group). There was a tendency (p =
0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (> I
ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1 %). When only cows that
were successfully synchronized were considered, the magnitude of the absolute difference in the
percentage of inseminated cows that were diagnosed pregnant between bST and control cows was
reduced (24.8 vs 22.4% pregnant for bST and control).
Conclusion: Results failed to indicate a beneficial effect of bST treatment on fertility of lactating
dairy cows.






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Background
Treatment of lactating cows with recombinant bovine
somatotropin (bST) can increase milk yield in thermone-
utral and heat-stress environments [1-6]. The conse-
quences of bST treatment for fertility are not clear. In early
studies, cows treated with bST had an increased calving to
conception interval that was attributed, at least in part, to
increased anestrus [7,8]. In contrast, bST either increased
[9,10] or had no effect [6,11,12] on pregnancy rate per
insemination when cows were inseminated using a timed
artificial insemination (TAI) protocol. Treatment with bST
increased pregnancy rate per insemination for repeat-
breeder cows bred at estrus [13].

Bovine ST might be particularly effective at increasing fer-
tility during periods of heat stress. This is because insulin-
like growth factor-1 (IGF-1), whose secretion is stimulated
by bST [6,14], has thermoprotective properties and can
reduce the effect of elevated temperature on development
and apoptosis of cultured bovine embryos [15,16].
Indeed, the proportion of heat-stressed lactating cows
receiving an in vitro produced embryo that became preg-
nant was higher when embryos were cultured in IGF-1
before transfer [17,18]. This effect of IGF-1 was not seen
for cows in cool weather [18].

Despite the promise of using bST in the summer to
increase IGF-1 and improve embryonic resistance to heat
stress, there was no effect of bST on pregnancy rate in the
one experiment that examined bST effects on fertility dur-
ing heat stress [6]. In this experiment, cows received bST
every 14 days beginning 13 days before TAI. The failure to
observe an increase in fertility caused by bST may have
been because bST also increased body temperature.

The hypothesis of the current study was that a single injec-
tion of a sustained-release preparation of bST during the
preovulatory period could improve fertility of lactating
cows. The rationale for the hypothesis was that bST,
directly or through an increase in IGF-1 secretion, would
increase fertility by exerting beneficial effects on follicular
function [19,20], oocyte function [21,22] and embryonic
development [23,24]. In heat-stressed cows, bST would


also be thermoprotective towards the embryo through
actions of IGF-1 [15,16]. Treatment with bST was limited
to a single injection around the time of ovulation to exert
effects on late follicular development, ovulation, and
early embryonic development while avoiding milk yield
responses during the peri-ovulatory period that could
enhance hyperthermia or otherwise limit the possible
beneficial effect of bST on fertility.

Results
Effects of bST at insemination in cows in a temperate
climate (Experiment I)
As shown in Table 1, there was no effect of bST on the pro-
portion of cows that were pregnant following insemina-
tion. In addition, there was no significant effect of
breeding at estrus vs TAI (Table 1), parity or days in milk
(results not shown) on pregnancy rate per insemination.

Effects of bST in cows exposed to heat stress (Experiment
2)
The 24-h pattern of vaginal temperatures is shown in Fig.
1. Time of day affected vaginal temperature (p < 0.001).
The peak temperature (least-squares mean) for both
groups was 39.3C and occurred at 23:00 h for control
cows and 18:00 h for bST-treated cows. The nadir, 38.6C
for both control and bST-treated cows, occurred at 08:00
and 09:00 h, respectively. Vaginal temperature was not
affected by bST treatment, day relative to TAI, or interac-
tions between these main effects and other variables.

Body condition score at TAI was not different between
control and bST-treated cows (2.52 0.03 vs 2.54 + 0.03).

Cows were considered to be successfully synchronized if
progesterone concentration on the day of TAI (Day 0) was
< 1 ng/ml and progesterone concentration on Day 7 after
TAI was > 1 ng/ml. Using this criterion, there was a ten-
dency (p = 0.100) for a lower percent of control cows to be
synchronized than bST-treated cows (77.3 vs 86.1%;
Table 2). Further examination of progesterone concentra-
tions revealed that, regardless of treatment, over 90% of
cows had low progesterone concentrations (< 1 ng/ml) at
Day 0. However, there was a tendency (p = 0.101) for a


Table I: Effect of bovine somatotropin administered at insemination on the proportion of cows inseminated at first service that
became pregnant (Experiment I).a
Treatment


Cows bred at estrus
Cows subject to TAI
Total


aAbbreviations are bST; bovine somatotropin; AOR, adjusted odds ratio; Cl, 95% confidence interval.
b Data are the number of cows pregnant/total cows inseminated and, in parentheses, percent pregnant.


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Control


20/65 (31%)
1 1/35 (31%)
31/100 (31%)


bST

19/65 (29%)
10/35 (29%)
29/100 (29%)


p AOR



NS 0.96


Cl



0.69-1.32


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39.4


39.2



39.0



38.8



38.6


3 8 .4 .. . I.I. I I I I I
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 192021 222324

Time of Day (h)

Figure I
Effect of bovine somatotropin on vaginal temperature (Experiment 2). Data represent least-squares means SEM.
There was an effect of time (p < 0.001) but no difference between control (blue circles) and bST-treated cows (red circles) and
no time x treatment interaction.


smaller percent of control cows to have high progesterone
(> 1 ng/ml) at Day 7 than for bST-treated cows (72.6 vs
81.1%; Table 2). Thus, a smaller percent of cows ovulated
in response to TAI for control cows than for bST-treated
cows.

Body condition score (BCS) at TAI also affected the suc-
cessful synchronization rate (p = 0.03). Cows with BCS _>
2.5 were more likely to have been successfully synchro-
nized (125/152, 87.5%) than cows with BCS < 2.5 (57/


80, 71.3%) [adjusted odds ratio (AOR) = 0.36, 95% con-
fidence interval (CI) = 0.18 0.71].

Results for pregnancy following TAI are presented in Table
3. Among all cows, the percent of cows pregnant follow-
ing TAI was not statistically affected by treatment. Numer-
ically, the percent of inseminated cows that was pregnant
was lower for control cows (17.8 %) than for cows treated
with bST (24.2%). This nonsignificant difference between
groups was diminished in magnitude when the percent of
inseminated cows that were pregnant was calculated for


Table 2: Effect of bovine somatotropin administered 3 days before insemination on plasma progesterone concentrations on Day 0 and
7 relative to timed artificial insemination and on the percent of cows successfully synchronized (Experiment 2).a

Treatment


p AOR Cl


Low progesterone (< I ng/ml) on Day 0
High progesterone (> I ng/ml) on Day 7
Synchronized (low on d 0 and high on Day 7)


112/124 (90.3%)
90/124 (72.6%)
85/110 (77.3%)


123/132 (93.2%)
107/132 (81.1%)
105/122 (86.1%)


NS 1.45 0.58 to 3.58
0.10 1.66 0.91 to 3.03
0.10 1.78 0.89 to 3.58


aAbbreviations are bST; bovine somatotropin; AOR, adjusted odds ratio; CI, 95% confidence interval; NS, non-significant
b Data are the number of cows with that classification/total cows inseminated and, in parentheses, percent.


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Control


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Table 3: Effect of bovine somatotropin administered 3 days before insemination on the proportion of cows pregnant after first service
(Experiment 2).a

Treatment


All cows
Successfully synchronized cows c


aAbbreviations are bST; bovine somatotropin; AOR, adjusted odds ratio; Cl, confidence interval; NS, non-significant
b Data are the number of cows pregnant/total cows inseminated and, in parentheses, percent pregnant.
cCows where progesterone concentration on the day of timed artificial insemination (Day 0) was < I ng/ml and progesterone concentration on Day
7 after TAI was > I ng/ml.


the subset of cows that were successfully synchronized
(22.4 vs 24.8%, treatment effect, p > 0.10).

Discussion
Results from the present study failed to support the
hypothesis that administration of a sustained-release
form of bST near the time of insemination improves fer-
tility of lactating dairy cows. The hypothesis that bST
improves fertility was tested independently at two differ-
ent locations, with two different commercial bST prepara-
tions and using different breeding procedures. In the first
experiment, conducted in the absence of heat stress and
with most cows bred at natural estrus, there was no signif-
icant effect of bST on the percent of cows pregnant follow-
ing first service. The preparation of bST used in the first
experiment also contained vitamin E. Administration of
vitamin E at insemination was without effect on preg-
nancy rate in lactating cows [25]. In the second experi-
ment, where cows were heat-stressed and where TAI was
the sole method for breeding cows, there was a numerical
increase in the percent of cows pregnant following insem-
ination for cows treated with bST as compared to control
cows. However, this difference was not statistically differ-
ent. Moreover, the numerical increase associated with bST
was associated with effects on the response to ovulation
synchronization rather than to effects on fertilization rate
or embryonic survival. Specifically, bST tended to
improve the percent of cows that ovulated after the ovula-
tion synchronization protocol. The difference in percent
of cows pregnant after TAI between control and bST-
treated cows was reduced when only successfully-synchro-
nized cows were considered.

Experiments to evaluate the beneficial effects of bST on
ability of cattle to establish and maintain pregnancy after
insemination in cattle have yielded variable results.
Administration of a single injection of bST at insemina-
tion did not improve pregnancy rate per insemination in
cows (largely beef cows) or beef heifers [26]. However, a
single injection of bST at estrus increased the percent of
repeat-breeder dairy cows pregnant following insemina-
tion [13]. Injections of bST increased pregnancy success in


dairy cows bred to TAI in some studies [9,10] but not in
others [ 6,11,12]. Differences in treatment regimens, prod-
uct formulation, reproductive management, cow type and
issues related to sample size could explain some of this
variation. For example, bST improved pregnancy success
for cows subjected to a Presynch-Ovsynch synchroniza-
tion protocol but did not cause improvement for cows
receiving an Ovsynch protocol without presynchroniza-
tion [10]. Taken together, the lack of a consistent fertility-
promoting effect of bST would contraindicate broad use
of bST to improve fertility in lactating cows. Additional
work is needed to confirm its efficacy in selected popula-
tions of cows, for example in repeat-breeder cows [13].

It was hypothesized that cows exposed to heat stress
would be particularly likely to benefit from administra-
tion of bST because IGF-1, which is released in response
to bST, can protect bovine preimplantation embryos from
the deleterious effects of elevated temperature on develop-
ment [13,14]. Nonetheless, bST did not improve fertility
in Experiment 2 despite the fact that cows experienced
vaginal temperatures characteristic of hyperthermia
(greater than 38.6 C) throughout most of the day. There
are several explanations for the lack of effect ofbST during
heat stress. For instance, it is possible that bST was not
administered until damage to the oocyte had already
occurred. Heat stress can compromise the follicle some-
where between 20-50 days before ovulation [27] and
administration of bST three days before ovulation, as per-
formed here, would probably not reverse oocyte damage
occurring earlier in follicular development. The bST was
not administered earlier relative to ovulation in Experi-
ment 2 because of the desire to avoid the effect of bST on
milk yield that could possibly result in increased body
temperature [5]. In fact, the bST treatment did not
increase vaginal temperature. Another possibility is that
early embryos are refractory to the thermoprotective
actions of IGF-1. The only stage of development at which
IGF-1 has been shown to reduce negative effects of ele-
vated temperature on embryos is at Day 5 after insemina-
tion [15,16]. Finally, it is possible that treatment with bST
may not change the bioavailable IGF-1 in the follicular,


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Control


22/124 (17.8%)
19/85 (22.4%)


p AOR


32/132 (24.2%)
26/105 (24.8%)


0.81 to 2.76
0.51 to 2.20


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oviductal or uterine environment sufficiently to protect
oocytes or embryos from elevated temperature. Treatment
of lactating cows with bST increased amounts of IGF-1 in
follicular fluid but not in uterine flushings [28].

In Experiment 2, the proportion of cows that ovulated fol-
lowing the ovulation synchronization protocol tended to
be greater for cows treated with bST. This possible effect of
bST did not reflect a difference in luteolysis following
administration of PGF20 because there was no difference
between control and bST-treated cows in the proportion
of cows with low progesterone concentrations on the
putative day of ovulation (Day 0). Perhaps bST increased
luteinizing hormone (LH) release in response to GnRH or
increased the rate of follicular development so that preo-
vulatory follicles were more likely to respond to LH. The
literature is equivocal as to whether bST affects LH secre-
tion [29,30] but it is well established that bST can enhance
follicular growth [18].

Conclusion
Results failed to indicate a beneficial effect of bST treat-
ment on fertility of lactating dairy cows.

Methods
Effect of bST at insemination in cows in a temperate
climate (Experiment I)
The study was carried out at an 1100-cow dairy farm
located in the central plateau of Mexico (Municipio de El
Marques, Estado de Queretaro, 20.31 N 100.09 W). The
climate is temperate and sub-humid, with rains during the
summer and an average yearly temperature of 18.7 C. The
study was conducted from July December 2006. The
average daily minimum and maximum dry bulb tempera-


tures and average daily relative humidities during the
study obtained from the Servicio Meteorol6gico Nacional
[31] for El Marques are shown in Table 4. Cows were
milked twice daily and the average milk production per
cow for the herd was 9800 kg per lactation. Cows were
kept in a free-stall barn and fed a total mixed ration six
times daily based on alfalfa hay, corn silage and concen-
trate according to National Research Council require-
ments [32].

The protocol used procedures routinely performed on the
dairy and approval from the local animal care committee
was not required. All cows eligible for first service were
considered for the study. Those diagnosed by a veterinar-
ian with retained placenta, pyometra, cystic ovaries or
endometritis or those with a BCS < 2.5 at artificial insem-
ination using a scale of 1 to 5 [33] were excluded. Cows
were paired according to insemination date and randomly
assigned within pair to receive either a single injection of
a sustained-release formulation of bST (n = 100; 500 mg,
s.c.; Boostin -S, Schering-Plough, Mexico; also contains
1.8 g vitamin E) or a control injection (n = 100; 2 ml of a
saline placebo) at first service. Injections were given by the
investigators at the same time as when insemination using
frozen-thawed semen was performed by a trained farm
technician.

Estrus was observed from Day 50 postpartum onwards by
direct observation continuously for 4-h periods in the
morning and the evening. At ~Day 60 postpartum (range
56 to 64 days), cows were palpated per rectum. Cows with
a corpus luteum were allowed to return to estrus naturally
(n = 130) and were inseminated 12 h after detection of
estrus. Cows without a corpus luteum or follicle > 10 mm


Table 4: Average daily minimum and maximum dry bulb temperatures and average daily relative humidity during the experiments.


Average daily minimum dry bulb
temperature (C)


Average maximum dry bulb temperature Average daily relative humidity
(C)


Experiment I (Mexico)

July
August
September
October
November
December

Experiment 2 (Florida)


June
July
August
September
October
November


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in diameter were considered anestrus and excluded from
the study. Cows without a corpus luteum and with a folli-
cle >10 mm in diameter were subjected to the Ovsynch
protocol (n = 70) [34]. Cows received gonadotropin
releasing hormone (GnRH, 100 rig, i.m.; Ovalyse, Pfizer,
Mexico, D.F., Mexico) in the morning. Seven days later, in
the morning, cows received a luteolytic dose of prostag-
landin F2, (PGF2c, 500 |ig i.m.; Celosil, Schering-Plough,
Xaltocan, Xochimilco, Mexico) in the morning. Two days
later, cows received a second injection of GnRH in the
afternoon and cows were inseminated 16 h later. The aver-
age days in milk at insemination was 69 + 14 days. Preg-
nancy diagnosis for cows not returning to estrus was
performed by rectal palpation at -45 days after insemina-
tion.

The proportion of inseminated cows that became preg-
nant at first service was analyzed by logistic regression
with JMP6 software (JMP, Release 6. 2005, SAS Institute
Inc., Cary, NC, USA). The analysis included effect of treat-
ment, type of estrus (spontaneous or synchronized), par-
ity (2 vs > 2) and days in milk (continuous).

Effects of bST at insemination for cows in hot conditions
(Experiment 2)
The experiment was conducted from June November
2006 at a commercial 3000-cow dairy in Trenton, Florida,
USA (29.360 N 82.490 W) utilizing 256 first-service, lac-
tating Holstein cows. The climate is semi-tropical and
humid. Meteorological measurements for the period of
the study obtained from the Florida Automated Weather
Network [35] for nearby Alachua, Florida are shown in
Table 4. Cows were housed in a free-stall barn equipped
with cooling fans and an automatic sprinkler system.
Cows were milked two times daily in the morning and
afternoon and fed as a group after each milking. The diet
was a total mixed ration containing corn silage and alfalfa
as the main ingredients with 77 Mcal of net energy for lac-
tation/kg and 17.4% crude protein on a dry matter basis.

The experimental protocol was approved by the Univer-
sity of Florida Institutional Animal Care and Use Com-
mittee Cows (project E730). Cows were selected for the
study because they were eligible for first service during the
study period. Exclusions in Experiment 1 were not consid-
ered. A presynchronization-OvSynch protocol was uti-
lized for first service TAI [36]. The protocol was initiated
at an average of 37 days postpartum (range 30 to 50). For
presynchronization, cows were administered 2 injections
of 25 mg PGF2, (Pfizer Animal Health, New York, NY,
USA, i.m.) at a 14-day interval. After another 14 days, an
ovulation synchronization protocol was initiated consist-
ing of 100 ig GnRH (Cystorelin, Merial Co., Athens, GA,
USA, i.m.) followed 7 days later with 25 mg PGF2, i.m.,
and a second injection of GnRH (100 jig) at 72 h follow-


ing PGF2, and coincident with TAI (at an average of 75
days postpartum). All injections were given in the morn-
ing. Several cows were observed in estrus and inseminated
prior to completion of the synchronization protocol.
These cows were excluded from the analysis. Cows
observed in standing estrus post-TAI were re-inseminated
and considered non-pregnant to the TAI. Pregnancy diag-
nosis for cows not returning into estrus was conducted by
rectal palpation at an average of 45 days after TAI.

Cows were enrolled in the experiment each week (during
a 12-week period during June September) and were
assigned randomly within each week to receive bST or
serve as controls. Treatment with a sustained-release for-
mulation of bST (500 mg, s.c.; Posilac, Monsanto Co., St
Louis, MO, USA) was performed at the same time as the
last PGF2,injection of the synchronization protocol.
Treatment was administered by the researchers in the
depression on either side of the tail head. Control cows
received no injection.

Body condition score was recorded for control and bST-
treated cows at insemination using a scale of 1 to 5 in 0.25
increments [33]. Each week, a random subset of cows in
each treatment was fitted with a temperature data logger
(HOBO Water Temp Pro vI, Onset Computer Co.,
Bourne, MA, USA) attached to a blank control internal
drug release insert (Pfizer Animal Health) that was
inserted into the vagina. The data logger recorded vaginal
temperature at 15-min intervals beginning at 1100 h on
Day 4 post-TAI for 3 days. Data were downloaded from
the data logger into a laptop computer after removal from
the cow. Average vaginal temperature for each hour was
calculated. A new subset of cows was enrolled each week
so that vaginal temperatures were recorded from a total of
21 control cows and 21 bST-treated cows. Note that
results of a previous experiment indicated that insertion
of HOBO devices into the vagina for 7 days did not affect
fertility [6].

Blood samples were collected from each cow at the time
of insemination and at Day 7 after insemination to deter-
mine plasma progesterone concentration. Blood samples
were collected by coccygeal venipuncture (artery or vein)
into evacuated heparinized 10-mL tubes (Becton Dickin-
son, Franklin Lakes, NJ, USA). Following collection,
blood samples were placed in an ice chest until further
processing in the laboratory (within approximately 2 to 6
h). Blood samples were centrifuged for 15 min at 4C at
3000 x g. Plasma was separated and stored at -20 oC until
assayed for progesterone concentration using a commer-
cial radioimmunoassay (Coat-a-Count, Diagnostic Prod-
ucts Corporation, Los Angeles, CA, USA). The intra- and
interassay coefficients of variance were 6% and 9%,
respectively, and assay sensitivity was 0.11 ng/ml. Cows


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with low plasma progesterone concentrations (< 1 ng/ml)
were considered to not have a functional corpus luteum
while cows with high plasma progesterone concentrations
(> 1 ng/ml) were considered to have a functional corpus
luteum. Cows that had low progesterone concentration
on Day 0 and high concentration on Day 7 were deter-
mined to be successfully synchronized in response to the
ovulation synchronization regimen.

Data on the proportion of cows pregnant after TAI were
analyzed with the LOGISTIC procedure of SAS using a
backward stepwise logistic model. Data were analyzed for
all cows and for the subset of cows that were successfully
synchronized. Variables were continuously removed from
the model by the Wald statistic criterion if the significance
was greater than 0.20. The initial mathematical model
included the effects of bST, sire, technician, month of
insemination, presence of a HOBO device, BCS class (<
2.5, > 2.5), bST x BCS class, and bST x month. The final
model included main effects of bST and month of insem-
ination. The AOR estimates and the 95% Wald confidence
intervals from logistic regression were obtained from this
analysis. The Wald chi-square statistic was used to deter-
mine the probability value for each main effect that
remained in the reduced model (significance was consid-
ered as p < 0.05).

Effects on the proportion of cows that were successfully
synchronized for TAI was also analyzed with the LOGIS-
TIC procedure of SAS using a backward stepwise logistic
model as described above. The final model included main
effects of bST and month of insemination.

Treatment effects on BCS and vaginal temperature were
analyzed using least-squares analysis of variance with the
GLM procedure of SAS. Tests of significance were made
using the appropriate error terms based on calculation of
expected means squares. Cow was a random effect and
other main effects were considered fixed. The model for
effects on BCS included month of insemination, bST and
the interaction. The model for effects on vaginal tempera-
ture included bST, cow (bST), day, time of day, and all two
way interactions.

Abbreviations
AOR: adjusted odds ratio; BCS: body condition score; bST:
bovine somatotropin; CI: 95% confidence interval;
GnRH: gonadotropin releasing hormone; IGF-1: insulin-
like growth factor-1; LH: luteinizing hormone; NS: non-
significant; PGF2c: prostaglandin F2,; TAI: timed artificial
insemination.


Competing interests
The authors received no financial support or donation of
materials from any commercial company. Moreover, the
authors have no competing interests.

Authors' contributions
All authors contributed to the experimental design and
interpretation of experiments. AB, OAR, LAdeC, MBP and
PJH administered treatments and collected data and JH-C,
CCG, PJH and ADV oversaw the projects.

Acknowledgements
Research was supported in part by grants from the US-Israel Binational
Agricultural Research and Development Fund (grant number US3986-07)
and Universidad Nacional Aut6noma de Mexico (IN223907). The authors
thank Hilltop Dairy and Ernie and Pam Bliss for their invaluable coopera-
tion.

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