Discovery and structure activity relationship of glyoxamide derivatives as anti-hepatitis B virus agents

Article abstract of DOI:10.1016/j.bmc.2020.115952

Chronic hepatitis B viral infection is a significant health problem world-wide, and currently available antiviral agents suppress HBV infections, but rarely cure this disease. It is presumed that antiviral agents that target the viral nuclear reservoir of transcriptionally active cccDNA may eliminate HBV infection. Through a series of chemical optimization, we identified a new series of glyoxamide derivatives affecting HBV nucleocapsid formation and cccDNA maintenance at low nanomolar levels. Among all the compounds synthesized, GLP-26 displays a major effect on HBV DNA, HBeAg secretion and cccDNA amplification. In addition, GLP-26 shows a promising pre-clinical profile and long-term effect on viral loads in a humanized mouse model.

Full text of DOI:10.1016/j.bmc.2020.115952

J. Dairy Sci. 97:6305–6315
http://dx.doi.org/10.3168/jds.2014-8222
®
©
American Dairy Science Association , 2014.
Presynchronization using a modified Ovsynch protocol
or a single gonadotropin-releasing hormone injection 7 d
before an Ovsynch-56 protocol for submission of lactating
dairy cows to first timed artificial insemination
1
P. D. Carvalho, J. N. Guenther, M. J. Fuenzalida, M. C. Amundson, M. C. Wiltbank, and P. M. Fricke
Department of Dairy Science, University of Wisconsin, Madison 53706
ABSTRACT
at first AI by increasing synchrony to the Ovsynch-56
protocol compared with presynchronization using a
single injection of GnRH.
Key words: presynchronization, Ovsynch, Double-
Ovsynch
Presynchronization strategies, such as Presynch-
Ovsynch and Double-Ovsynch, increase fertility to
timed artificial insemination (TAI) compared with
Ovsynch alone; however, simpler presynchronization
strategies could reduce costs and simplify reproduc-
tive management. Lactating Holstein cows (n = 601)
were randomly assigned to 1 of 2 presynchronization
treatments before beginning an Ovsynch-56 protocol
INTRODUCTION
Despite improvements in nutrition, cow comfort, and
genetic selection for reproductive efficiency that have
occurred over the past decade, pregnancies per artificial
insemination (P/AI) continues to be low (~ꢀ0%) in
high-producing dairy cows inseminated after a detected
estrus (Valenza et al., 2012; ꢂricke et al., 2014). Ex-
pression of behavioral estrus is poor in high-producing
dairy cows (Lopez et al., 2004) and ranged from 50%
after visual observation (Washburn et al., 2002) to 70%
when insemination was based on an activity monitoring
system for detection of estrus (Valenza et al., 2012), and
inadequate and inaccurate detection of estrus continues
to be a challenge on dairy farms (Palmer et al., 2010).
Timed artificial insemination (TAI) programs, such as
the Ovsynch protocol (Pursley et al., 1995), have been
developed and widely adopted by commercial dairies
(
GnRH at 70 ± ꢀ DIꢁ, PGꢂ2α 7 d later, GnRH 56
h after PGꢂ , and TAI 16 h later at 80 ± ꢀ DIꢁ)
2α
for first TAI. Cows (n = ꢀ06) in the first treatment
Double-Ovsynch; DO) were presynchronized using a
(
modified Ovsynch protocol (GnRH at 5ꢀ ± ꢀ DIꢁ, 7 d
later PGꢂ , and GnRH ꢀ d later) ending 7 d before the
2α
first GnRH injection (G1) of an Ovsynch-56 protocol.
Cows (n = 295) in the second treatment (GGPG) were
presynchronized using a single injection of GnRH 7 d
before G1 of an Ovsynch-56 protocol at 6ꢀ ± ꢀ DIꢁ.
Blood samples were collected at G1 and the PGꢂ2α
injections of the Ovsynch-56 protocol to determine
progesterone (P4) concentrations. Pregnancy diagnosis
was performed using ultrasonography ꢀ2 d after TAI,
and pregnant cows were reexamined 46 and 70 d af-
ter TAI. Overall, DO cows had more pregnancies per
artificial insemination (P/AI) compared with GGPG
cows ꢀ2 d after TAI (5ꢀ vs. 4ꢀ%). Overall, P/AI did
not differ by parity (primiparous vs. multiparous), and
pregnancy loss did not differ between treatments or
parities. ꢁore DO cows had P4 in a medium range
(
Caraviello et al., 2006). The implementation of syn-
chronization protocols that allow for TAI increases the
rate at which cows are inseminated after calving and,
in some situations, results in more P/AI compared with
cows inseminated to a detected estrus (Pursley et al.,
1
997; Gumen et al., 2012; ꢂricke et al., 2014).
Presynchronizationstrategiesthatincreasefertilityby
optimizing the hormonal milieu, in which the ovulatory
follicle grows, have been developed. Presynchronization
strategies have used 2 injections of PGꢂ2α administered
(
>0.5 to <4 ng/mL) at G1 of the Ovsynch-56 protocol
compared with GGPG cows (82 vs. 50%), and more
DO cows had high P4 (>4 ng/mL) at the PGꢂ2α injec-
tion of the Ovsynch-56 protocol compared with GGPG
cows (67 vs. ꢀ6%). Thus, presynchronization with a
modified Ovsynch protocol increased P/AI after TAI
1
4 d apart, with the second injection administered 10
to 14 d before initiation of an Ovsynch protocol (i.e.,
Presynch; ꢁoreira et al., 2001; Navanukraw et al.,
2
004), a single GnRH injection 7 d before Ovsynch [i.e.,
GnRH-GnRH-PGꢂ -GnRH (GGPG); Lopes et al.,
2
α
2
01ꢀ; Bruno et al., 2014], and a combination of GnRH
Received April 10, 2014.
Accepted June 2ꢀ, 2014.
and PGꢂ2α 6 to 7 d before initiation of an Ovsynch
protocol [i.e., G-6-G and Double-Ovsynch (DO); Bello
1
Corresponding author: pmfricke@wisc.edu
6305

6306
CARVALHO ET AL.
et al., 2006; Souza et al., 2008]. Independent of the of Agriculture and Life Sciences at the University of
presynchronization strategy used (Double-Ovsynch, Wisconsin-ꢁadison.
Presynch-Ovsynch, G-6-G), an improvement in fertility
was reported when progesterone (P4) concentrations
were increased at the time of the PGꢂ2α injection of
an Ovsynch protocol (Bello et al., 2006; Bisinotto et
Cows, Housing, and Feeding
The current study was conducted at the University
al., 2010; ꢁartins et al., 2011; Denicol et al., 2012). of Wisconsin-ꢁadison Emmons Blaine Dairy Cattle
Although presynchronization strategies have led to dra- Research Center (Arlington, WI) from ꢁay 2012 to
matic improvements in P/AI compared with AI after August 201ꢀ. Holstein cows (n = 601) were milked
a detected estrus or an Ovsynch protocol alone, they twice daily approximately 12 h apart and were fed once
have increased or doubled the number of sequential daily a TꢁR consisting of corn and alfalfa silage as
injections required for a cow to complete the protocol. forage with corn and soybean meal-based concentrate
Simplification of hormonal protocols for TAI may im- formulated to meet or exceed the minimum nutritional
prove compliance and reduce costs and labor associated requirements for high-producing dairy cows (NRC,
with reproductive management. Presynchronization of 2001). Cows were housed in freestall barns bedded with
cows receiving second and greater TAI using a single in- sand and had ad libitum access to feed and water. Pri-
jection of GnRH administered 7 d before initiation of an miparous cows were housed in separate pens from mul-
Ovsynch-56 protocol (i.e., a GGPG protocol) improved tiparous cows. Barns were equipped with fans, but not
fertility compared with Ovsynch alone (Dewey et al., sprinklers, that were automatically activated when the
2
010; Lopes et al., 201ꢀ; Bruno et al., 2014). Although temperature inside the barns reached 68°ꢂ (20°C). As
a direct comparison of a Double-Ovsynch protocol to a per the management procedures in place at the dairy,
GGPG protocol has not been reported, comparison of multiparous cows received somatotropin (500 mg/dose;
presynchronization with a single human chorionic go- Posilac, ꢁonsanto Co., St Louis, ꢁO) every 14 d start-
nadotropin treatment 7d before Ovsynch (hGPG) dur- ing at 67 ± ꢀ d postpartum, whereas somatotropin was
ing a Resynch program yielded similar P/AI compared not administered to primiparous cows. The rolling herd
with cows resynchronized using a Double-Ovsynch average was 1ꢀ,880 kg and average daily milk produc-
protocol (Giordano et al., 2012b). A subsequent study tion was 41.ꢀ kg/cow per day with ꢀ.6% fat and ꢀ.1%
comparing strategies for resynchronization showed no protein for the entire herd during the time period of
statistical difference between P/AI after an hGPG pro- this experiment.
tocol compared with a GGPG protocol (Giordano et
al., 2012b). ꢂor submission of cows for first TAI, the
Double-Ovsynch protocol has been directly compared
with a Presynch-Ovsynch protocol (Souza et al., 2008;
Treatments and AI
Every week, cows from 5ꢀ ± ꢀ DIꢁ were blocked
Herlihy et al., 2012; Ribeiro et al., 2012); however, a di- by parity (primiparous vs. multiparous) and were
rect comparison between the Double-Ovsynch protocol randomly assigned to have their estrous cycle presyn-
and a simpler GGPG protocol for submission of cows chronized using either a modified Ovsynch protocol or
for first TAI has not been reported.
a single GnRH injection administered 7 d before an
Therefore, the aim of the current study was to com- Ovsynch-56 protocol to receive their first TAI (ꢂigure
pare presynchronization using a modified Ovsynch pro- 1). The 2 presynchronization schemes resulted in a DO
tocol (i.e., Double-Ovsynch) or a single GnRH injection protocol (Souza et al., 2008; Herlihy et al., 2012) and
administered 7 d before initiation of an Ovsynch-56 a GGPG protocol (Giordano et al., 2012b; Lopes et
protocol for submission of lactating dairy cows for first al., 201ꢀ), respectively. Prostaglandin ꢂ2α (25 mg of
TAI (i.e., GGPG). We further wanted to determine the dinoprost tromethamine; Lutalyse) was from Zoetis
effect of these presynchronization strategies on P4 con- (New York, NY). The GnRH (100 μg of Gonadorelin
centrations during and after the Ovsynch-56 portion of diacetate tetrahydrate; Cystorelin) was from ꢁerial
the protocols. Based on data from resynchronized cows (Duluth, GA). Cows (n = ꢀ06) in the DO treatment
(
Giordano et al., 2012b), our hypothesis was that sub- received the first GnRH injection of the Presynch por-
mission of cows for first TAI using a GGPG protocol tion of the DO protocol at 5ꢀ ± ꢀ DIꢁ, followed by
would result in similar fertility to cows receiving first an injection of PGꢂ2α 7 d later and GnRH 72 h after
TAI after a Double-Ovsynch protocol.
PGꢂ , and then began the Ovsynch-56 protocol 7 d
2α
later. Cows (n = 295) in the GGPG treatment received
a GnRH injection at 6ꢀ ± ꢀ DIꢁ, and then began the
MATERIALS AND METHODS
All animal handling and procedures were approved Ovsynch-56 protocol 7 d later. All cows received the
by the Animal Care and Use Committee of the College same Ovsynch-56 protocol [first GnRH (G1) at 70 ±
Journal of Dairy Science Vol. 97 No. 10, 2014

METHODS OF TIMED ARTIFICIAL INSEMINATION
6307
Figure 1. Schematic representation of the synchronization protocols. Double-Ovsynch cows received the first GnRH injection of the Presynch
portion of the Double-Ovsynch protocol at 5ꢀ ± ꢀ DIꢁ, followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after PGꢂ2α, then began an
Ovsynch-56 protocol 7 d later. The GGPG cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ and then began the Ovsynch-56 protocol 7 d later.
All cows received a similar Ovsynch-56 protocol: at 70 ± ꢀ d cows received a GnRH injection, followed by an injection of PGꢂ2α 7 d later and
GnRH 56 h after PGꢂ2α, and were given a timed AI (TAI) 16 to 20 h after the last GnRH injection. B = blood sample.
ꢀ
DIꢁ, PGꢂ2α 7 d later, GnRH 56 h after PGꢂ , and GGPG, n = 244) injections of the Ovsynch-56 protocol,
2α
AI 16 to 20 h later] and received TAI at 80 ± ꢀ DIꢁ. as well as at 4 (DO, n = 170; GGPG, n = 156) and
Three experienced AI technicians that were blinded to 11 d (DO, n = 171; GGPG, n = 156; ꢂigure 1) after
treatments performed all inseminations. ꢁultiple sires TAI. After collection, blood samples were refrigerated
with high genetic merit and proven fertility were used for 24 h and centrifuged (1,600 × g; 4°C) for 20 min.
and were equally balanced between treatments.
Serum was harvested and stored at −20°C until assayed
for P4 concentration using a solid-phase, no-extraction
radioimmunoassay (Coat-a-Count, Diagnostic Products
Corp., Los Angeles, CA). The average sensitivity for
Pregnancy Diagnosis
Pregnancy diagnosis was performed ꢀ2 d after TAI the 4 assays was 0.0ꢀ5 ng/mL. The average intraassay
for all cows in both treatments using a portable scan- CV was 4.04% and the interassay CV was 4.ꢀ2% based
ner (Ibex Pro, E. I. ꢁedical Imaging, Loveland, CO) on a quality control sample (2.50 ng/mL of P4), which
equipped with a 7.5-ꢁHz linear-array transducer. A was replicated within each assay.
positive pregnancy diagnosis was based on visualiza-
tion of a corpus luteum on the ovary ipsilateral to the
uterine horn containing an embryo with a heartbeat.
Pregnancy status for cows diagnosed pregnant was
BCS Evaluation and Milk Yield
Body condition score of a subgroup of cows (DO, n
reconfirmed at 46 and 70 d after TAI using the same = 228; GGPG, n = 218) was evaluated on the day of
ultrasound machine. Twin pregnancy diagnosis was the PGꢂ2α injection of the Ovsynch-56 protocol (77 ± ꢀ
performed on the day of the third pregnancy diagnosis DIꢁ) using a 5-point scale with 0.25 increments where
at 70 d after AI. Cows diagnosed pregnant and then 1 = thin and 5 = fat (Edmonson et al., 1989). The same
diagnosed not pregnant at the subsequent pregnancy individual performed all BCS evaluations throughout
examination were considered to have undergone preg- the experiment. ꢂor statistical analysis, BCS was cat-
nancy loss.
egorized as either low (≤2.50) or high (≥2.75).
ilk weights were recorded at each milking and
stored in the on-farm computer software program
DairyComp ꢀ05, Valley Agricultural Software, Tulare,
ꢁ
Blood Sampling and Progesterone Assay
(
Blood samples from a subgroup of cows were col- CA). Average weekly milk weights were extracted from
lected via puncture of the median caudal vein or artery the software program and used to calculate milk pro-
into 8-mL evacuated serum collection tubes (Vacuette, duction from calving to the first pregnancy diagnosis.
Greiner Bio-One North America Inc., ꢁonroe, NC). ꢂor statistical analysis, cows were categorized by parity
Samples were collected immediately before G1 (DO, n (primiparous vs. multiparous) according to milk pro-
=
255; GGPG, n = 244) and the PGꢂ2α (DO, n = 256; duction as being below or above mean milk production.
Journal of Dairy Science Vol. 97 No. 10, 2014

6308
CARVALHO ET AL.
Temperature and Humidity Recording
and Heat Stress
and season, as well as the interactions between these
variables, whereas cow was included as a random ef-
fect. Based on a covariance parameter estimate test,
the random effect of cow was removed from the model
so that all final models contained only fixed effects.
The covariance parameter test using the ZeroG option
for the Glimmix procedure was used to evaluate if the
matrix containing random effects could be reduced to
zero. A covariance parameter test based on the resid-
ual pseudolikelihood was run and a nonsignificant chi-
squared P-value indicated that random effects could
be eliminated from the model (SAS Documentation for
GLIꢁꢁIX, SAS 9.ꢀ). After removing random effects,
selection of the fixed effects model that best fit the data
for each variable of interest was performed by finding
the model with the lowest value for the Akaike informa-
tion criterion using a backward elimination procedure
that removed all variables with a P-value exceeding
Temperature (°C) and relative humidity (RH; %)
data were obtained from the University of Wisconsin
Extension Ag Weather website (http://www.soils.wisc.
edu/uwex_agwx/). Weather data were collected from
ꢁ
ay 2012 to August 201ꢀ. The weather station is locat-
ed in Arlington, Wisconsin, approximately 0.5 km from
the dairy. ꢂor each 24-h period, average daily mean
and maximum temperature and RH were recorded and
a temperature humidity index (THI) was calculated.
Ambient temperature was converted from Celsius to
ꢂahrenheit [temperature in °ꢂ = (temperature in °C ×
1
.8) + ꢀ2], and the average and maximum daily THI
was calculated as THI = td – [0.55 – (0.55 × RH /100)]
td – 58), in which td is the temperature (in °ꢂ) and
(
RH is expressed as a percentage (NOAA, 1976). The
THI was averaged over the 10-d period preceding the
day of AI (from G1 to the day of TAI), and was the
criterion used to determine the season in which the
insemination occurred. Inseminations performed when
the average maximum THI was <72 were considered to
have occurred during the cool season, whereas insemi-
nations performed when the average maximum THI
was ≥72 were considered to have occurred during the
warm season. ꢂor statistical analysis, cows inseminated
when the average of maximum THI was >72 during the
0
.10 from the model. Both treatment and parity were
forced to remain in each model. Parity was forced into
the final model because it was used as a blocking factor
for randomization of cows to treatments. Therefore, for
P/AI at ꢀ2, 46, and 70 d after TAI, the final model
contained the fixed effects of treatment and parity.
Because season and parity were not significant in the
logistic regression model, differences in P/AI between
parities within treatments, as well as between seasons
within treatments, were obtained using a chi square
test.
1
0-d period before AI (from G1 to the day of TAI) were
classified as being exposed to heat stress, whereas cows
in which the average maximum THI was ≤72 during
the 10-d period before AI were classified as not being
exposed to heat stress.
The same categorical variables and interactions used
for P/AI were used to obtain the models for analysis of
pregnancy loss (from ꢀ2 to 46, 46 to 70, and from ꢀ2 to
7
0 d after TAI) and twin pregnancies. The procedures
and criteria used for model selection were similar to
those used for P/AI. The final model included the fixed
effects of treatment and parity.
Statistical Analyses
The experimental design was a complete randomized
Treatment differences in circulating P4 concentra-
block design with parity (primiparous vs. multiparous) tions at G1 and the PGꢂ2α injection of the Ovsynch-56
as the blocking factor. All statistical analyses were per- protocol, as well as at 4 and 11 d after TAI, were de-
formed using SAS computational software, version 9.ꢀ termined by ANOVA using the ꢁIXED procedure of
for ꢁicrosoft Windows (SAS Institute Inc., Cary, NC). SAS. The model contained as fixed effects treatment,
Analysis of binary response data (P/AI, pregnancy parity, and their interaction, whereas cow was used as
loss, and twins) was performed by logistic regression a random effect in the model. Data were examined for
using the GLIꢁꢁIX procedure of SAS. ꢂor P/AI at normality using the Shapiro-Wilk test. A significant P-
ꢀ
2, 46, and 70 d after TAI, the initial model contained value indicated that data were not normally distributed;
the following categorical explanatory variables as fixed therefore data were transformed to ranks. Cows were
effects: parity (primiparous vs. multiparous), treat- distributed into 9 categories using P4 concentration at
ment (DO vs. GGPG), level of milk production (high G1 and the PGꢂ2α injection of the Ovsynch-56 protocol
vs. low), BCS (≤2.5 vs. >2.5), and season (warm vs. (from 0.00 to ≥4.00 ng/mL in 0.5-ng/mL increments).
cool), as well as the interactions between these vari- At 11 d after TAI, cows were divided into 7 catego-
ables.
ries (0.00 to ≥7.00 ng/mL in 1.00-ng/mL increments).
All initial models contained both fixed and random Differences in the proportion of cows within each P4
effects. ꢂixed effects included in the initial models category were analyzed by logistic regression using the
were treatment, parity, level of milk production, BCS, LOGISTIC procedure of SAS. The effect of P4 on P/AI
Journal of Dairy Science Vol. 97 No. 10, 2014

METHODS OF TIMED ARTIFICIAL INSEMINATION
6309
was assessed by logistic regression using the LOGISTIC not differ (P = 0.ꢀ5) between treatments for primipa-
procedure of SAS with a model that only contained the rous cows, whereas multiparous GGPG cows had fewer
variable P4 concentration as fixed effect. ꢂor evalua- (P = 0.02) P/AI compared with multiparous DO cows
tion of the effect of P4 at the PGꢂ2α injection of the (Table 2). Pregnancies per AI at ꢀ2 d after TAI did not
Ovsynch-56 protocol on P/AI, cows with P4 concentra- differ (P = 0.15) between cows receiving AI during the
tions <1.00 ng/mL (n = 58) were removed from the cool and warm seasons [50 (204/412) vs. 44% (8ꢀ/189),
analysis, whereas cows with P4 concentrations >10.0 respectively]; however, P/AI at ꢀ2 d after TAI did not
ng/mL were classified as having P4 concentrations of differ (P = 0.88) for DO cows receiving TAI during the
1
0 ng/mL. The effect of milk production during the cool and warm season, whereas GGPG cows tended to
week before AI on P/AI was also evaluated by logis- have fewer (P = 0.07) P/AI after TAI during the warm
tic regression using the LOGISTIC procedure of SAS compared with the cool season (Table 2). Overall, P/AI
with a model that contained the fixed effects of milk did not differ (P = 0.16) between cows with high ver-
production on the week before AI (continuous), parity sus low BCS near TAI [48 (201/418) vs. ꢀ6% (10/28),
(
primiparous vs. multiparous), and their interaction.
Differences in milk production and BCS were deter-
respectively].
At 46 d after TAI, DO cows had more (P = 0.0ꢀ)
mined by ANOVA using the ꢁIXED procedure of SAS. P/AI than GGPG cows (Table 1). Overall, P/AI at
The model contained as fixed effects treatment, week 46 d after TAI did not differ (P = 0.17) between pri-
postpartum (for evaluation of milk production only), miparous and multiparous cows [50 (112/224) vs. 44%
parity, and their interaction, whereas cow was used as (167/ꢀ77), respectively]. At 70 d after TAI, DO cows
a random effect in the model.
had more (P = 0.05) P/AI than GGPG cows (Table 1).
A significant difference between the levels of a classi- Nevertheless, P/AI at 70 d after TAI did not differ (P
fication variable was considered when P ≤ 0.05, where- = 0.1ꢀ) between primiparous and multiparous cows [49
as differences between P > 0.05 and P ≤ 0.10 were (109/224) vs. 42% (160/ꢀ77), respectively].
considered a statistical tendency. Data are presented
Overall pregnancy loss for all cows from ꢀ2 to 46, 46
as means ± SEꢁ, obtained using ꢁEANS procedure to 70, and total loss from ꢀ2 to 70 d after TAI was 2.8,
of SAS.
ꢀ.6, and 6.ꢀ, respectively. Pregnancy loss from ꢀ2 to 46
d after TAI did not differ between treatments (Table
RESULTS
1
), parities (P = 0.92; 2.6 vs. 2.9%, for primiparous
vs. multiparous cows, respectively), or season (P =
.7ꢀ; 2.9 vs. 2.5%, for TAI during the cool vs. warm
season, respectively). Similarly, pregnancy loss from
6 to 70 d after TAI did not differ between treat-
Milk Production and BCS
0
The average milk yield from calving to the first
pregnancy diagnosis after first TAI did not differ (P =
4
0
.70) between DO and GGPG cows (47.1 vs. 46.7 kg/d,
ments (Table 1) or parities (P = 0.52; 2.7 vs. 4.2%
for primiparous vs. multiparous cows, respectively).
Overall pregnancy loss from ꢀ2 to 70 d after TAI did
not differ between treatments (Table 1) or parities (P
respectively). In addition, milk production did not dif-
fer (P = 0.71) between DO and GGPG cows (50.1 vs.
4
ꢁ
9.7 kg/d, respectively) during the week before TAI.
ilk production from calving to the first pregnancy
=
0.58; 5.2 vs. 7.0% for primiparous vs. multiparous
diagnosis after first TAI was greater (P < 0.001) for
multiparous cows compared with primiparous cows
cows, respectively).
Percentage of cows diagnosed with twins at 70 d
after TAI did not differ (P = 0.55) between DO vs.
GGPG cows (4.9 vs. 6.2%, for, respectively); however,
multiparous cows tended (P = 0.06) to have more twin
pregnancies compared with primiparous cows (7.8 vs.
(
5ꢀ.ꢀ vs. ꢀ6.0 kg/d, respectively). ꢂor the subgroup
of cows (n = 446) with BCS evaluated at the PGꢂ2α
injection of the Ovsynch-56 protocol, BCS did not dif-
fer (P = 0.68) between DO and GGPG cows (ꢀ.08 vs.
ꢀ
.09, respectively); however, primiparous cows had a
1
.9%, respectively). Overall, the percentage of cows
greater (P < 0.001) BCS compared with multiparous
diagnosed with twins at 70 d after TAI was 5.4%.
cows (ꢀ.21 vs. ꢀ.04, respectively).
P4 Concentrations
P/AI, Pregnancy Loss, and Twins
At G1 of the Ovsynch-56 protocol, mean P4 con-
At ꢀ2 d after TAI, DO cows had more (P = 0.01) centrations did not differ (P = 0.71) between DO and
P/AI than GGPG cows (Table 1). Nevertheless, P/AI GGPG cows (2.61 vs. ꢀ.11 ng/mL, respectively), and
at ꢀ2 d after TAI did not differ (P = 0.16) between did not differ (P = 0.16) between parities (ꢀ.02 vs.
primiparous and multiparous cows [51 (115/224) vs. 2.79 ng/mL, for primiparous vs. multiparous cows, re-
4
6% (172/ꢀ77), respectively]. ꢂurthermore, P/AI did spectively). When cows were divided into 9 categories
Journal of Dairy Science Vol. 97 No. 10, 2014

6310
CARVALHO ET AL.
Table 1. Effect of treatment on pregnancies per artificial insemination (P/AI) after first timed AI and
pregnancy loss in lactating Holstein cows
1
Treatment
Item
Double-Ovsynch
GGPG
P-value
P/AI, % (n/n)
ꢀ
4
7
2 d
6 d
0 d
52.6 (161/ꢀ06)
50.7 (155/ꢀ06)
48.7 (149/ꢀ06)
42.7 (126/295)
42.0 (124/295)
40.7 (120/295)
0.01
0.0ꢀ
0.05
Pregnancy loss, % (n/n)
ꢀ2 to 46 d
46 to 70 d
ꢀ2 to 70 d
ꢀ.7 (6/161)
ꢀ.9 (6/155)
7.5 (12/161)
1.6 (2/126)
ꢀ.2 (4/124)
4.8 (6/126)
0.29
0.80
0.ꢀ7
1
Double-Ovsynch cows received the first GnRH injection of the Presynch portion of the Double-Ovsynch pro-
tocol at 5ꢀ ± ꢀ DIꢁ, followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after PGꢂ2α, then began an
Ovsynch-56 protocol 7 d later. The GGPG cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ and then began
the Ovsynch-56 protocol 7 d later.
based on P4 concentrations at G1 (ꢂigure 2), more (P for cows with P4 concentrations of 0 to 1.0, 1.0 to 4.0,
0.01) DO cows had medium P4 concentrations (0.50 and >7 ng/mL, whereas P/AI were less for GGPG cows
to ꢀ.99 ng/mL) compared with GGPG cows (82 vs. with P4 concentrations between 4 and 7 ng/mL (Table
0%). In addition, fewer DO cows had low (<0.5 ng/ ꢀ). ꢂurthermore, when cows from both treatments were
mL) P4 concentrations (ꢀ vs. 17%; P < 0.01) or high combined to evaluate the effect of P4 concentration on
≥4.00 ng/mL) P4 concentrations (15 vs. ꢀꢀ%; P < P/AI (Table ꢀ), P/AI differed (P < 0.01) among P4
.01) compared with GGPG cows. categories and was least for cows with P4 concentra-
At the PGꢂ2α injection of the Ovsynch-56 protocol, tions of 0 to 1.0 ng/mL and greatest for cows with P4
<
5
(
0
mean P4 concentrations were greater (P < 0.001) for concentrations from 1.0 to 4.0, 4.0 to 7, and >7 ng/mL
DO cows compared with GGPG cows (5.ꢀ5 vs. ꢀ.55 (27.6, 47.ꢀ, 46.0, and 60.2%, respectively).
ng/mL, respectively), whereas mean P4 concentra-
At 4 d after TAI, mean P4 concentrations did not
tions were similar (P = 0.75) between primiparous and differ (P = 0.17) between DO vs. GGPG cows (0.89
multiparous cows (4.47 vs. 4.48 ng/mL, respectively). vs. 1.09 ng/mL, respectively); however, mean P4 con-
When cows were divided into 9 categories based on P4 centrations were greater (P = 0.01) for primiparous
concentrations at the PGꢂ injection of the Ovsynch-56 compared with multiparous cows (1.1ꢀ vs. 0.9ꢀ ng/
2
α
protocol (ꢂigure ꢀ), more (P < 0.01) DO cows had high mL, respectively). In addition, the distribution of cows
P4 (>4.0 ng/mL) compared with GGPG cows (67 vs. based on P4 concentrations 4 d after TAI was similar
ꢀ
6%), whereas more (P < 0.01) GGPG cows had lower between treatments (ꢂigure 4). When the effect of P4
P4 concentrations (0 to 1.99 ng/mL) than DO cows concentrations 4 d after TAI was evaluated, cows with
ꢀ9.8 vs. 7%). ꢂurthermore, more GGPG cows had P4 extremely low [P4 = <0.ꢀ ng/mL; 1ꢀ.0% (ꢀ/2ꢀ)] or
(
concentrations from 0 to 1.0 and 1.0 to 4.0 ng/mL, high [P4 = ≥2.0 ng/mL; 10.ꢀ% (ꢀ/29)] P4 had fewer
whereas more DO cows had P4 concentrations from 4 (P < 0.001) P/AI compared with cows with medium
to 7 and >7 ng/mL (Table ꢀ). ꢂor these ranges of P4 P4 concentrations [0.ꢀ ≥ P4 < 2.0 ng/mL; 50.7%
concentrations, P/AI were similar between treatments (1ꢀ9/274)].
Table 2. Effect of parity and season on pregnancies per AI (P/AI) at ꢀ2 d after first timed AI for lactating
1
Holstein cows synchronized using a Double-Ovsynch protocol or a GGPG protocol
Item, P/AI, % (n/n)
Double-Ovsynch
GGPG
P-value
Parity
Primiparous
54.5 (61/112)
51.5 (100/194)
0.62
48.2 (54/112)
ꢀ9.ꢀ (72/18ꢀ)
0.14
0.ꢀ5
0.02
ꢁultiparous
P-value
Season
Warm
52.0 (52/100)
52.9 (109/206)
0.88
ꢀ4.8 (ꢀ1/89)
46.1 (95/206)
0.07
0.02
0.17
Cool
P-value
1
Double-Ovsynch cows received the first GnRH injection of the Presynch portion of the Double-Ovsynch pro-
tocol at 5ꢀ ± ꢀ DIꢁ, followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after PGꢂ2α, then began an
Ovsynch-56 protocol 7 d later. The GGPG cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ, and then began
the Ovsynch-56 protocol 7 d later.
Journal of Dairy Science Vol. 97 No. 10, 2014

METHODS OF TIMED ARTIFICIAL INSEMINATION
6311
Figure 2. Distribution of cows based on progesterone concentra-
Figure 3. Distribution of cows based on progesterone concentra-
tions at the first GnRH injection (G1) of an Ovsynch-56 protocol for tions at the PGꢂ2α injection of an Ovsynch-56 protocol for cows pre-
cows presynchronized using a single GnRH injection (GGPG; black synchronized using a single GnRH injection (GGPG; black bars) or
bars) or a modified Ovsynch protocol (Double-Ovsynch; white bars) a modified Ovsynch protocol (Double-Ovsynch; white bars) for first
for first timed AI. Double-Ovsynch cows received the first GnRH injec- timed AI. Double-Ovsynch cows received the first GnRH injection of
tion of the Presynch portion of the Double-Ovsynch protocol at 5ꢀ ± ꢀ the Presynch portion of the Double-Ovsynch protocol at 5ꢀ ± ꢀ DIꢁ,
DIꢁ, followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after
PGꢂ2α, then began an Ovsynch-56 protocol 7 d later, whereas GGPG PGꢂ2α, then began an Ovsynch-56 protocol 7 d later, whereas GGPG
cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ and then began the cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ and then began the
Ovsynch-56 protocol 7 d later. All cows received a similar Ovsynch-56 Ovsynch-56 protocol 7 d later. All cows received a similar Ovsynch-56
protocol: at 70 ± ꢀ d cows received a GnRH injection, followed by an protocol: at 70 ± ꢀ d cows received a GnRH injection, followed by an
injection of PGꢂ2α 7 d later and GnRH 56 h after PGꢂ2α, and were injection of PGꢂ2α 7 d later and GnRH 56 h after PGꢂ2α, and were
given a timed AI 16 to 20 h after the last GnRH injection.
given a timed AI 16 to 20 h after the last GnRH injection.
At 11 d after TAI, mean P4 concentrations did not [12 (5/4ꢀ) vs. 50% (141/284)]; however, P/AI did not
differ (P = 0.19) between DO versus GGPG cows (ꢀ.85 differ (P = 0.75) among cows with P4 concentrations
vs. 4.07 ng/mL, respectively); however, mean P4 con- of 2 to ꢀ, ꢀ to 4, and >4 ng/mL [5ꢀ.ꢀ (ꢀ2/60) vs. 50.7
centrations were greater (P = 0.006) for primiparous (ꢀ5/69) vs. 47.7% (74/155), respectively].
compared with multiparous cows (4.52 vs. ꢀ.74 ng/mL,
respectively). The distribution of cows according to P4
concentrations 11 d after TAI did not differ between
DO versus GGPG cows (ꢂigure 5). Cows with P4 con-
DISCUSSION
Cows submitted for first TAI after a Double-Ovsynch
centrations <2 ng/mL had fewer (P < 0.001) P/AI protocol had more P/AI compared with cows submit-
compared with cows with P4 concentrations >2 ng/mL ted for first TAI using a Presynch-Ovsynch protocol in
1
Table 3. Effect of treatment on distribution of cows according to progesterone concentrations at the PGꢂ2α
injection of the Ovsynch-56 protocol and pregnancies per artificial insemination (P/AI) after first timed AI
Progesterone (ng/mL) at PGꢂ2α
Item
0.0 to 1.0
>1.0 to 4.0
>4.0 to 7.0
>7.0
Proportion of cows, % (n/n)
Double-Ovsynch
GGPG
ꢀ.1 (8/256)
20.5 (50/244)
<0.01
ꢀ0.9 (79/256)
4ꢀ.9 (107/244)
<0.01
4ꢀ.0 (110/256)
21.7 (5ꢀ/244)
<0.01
2ꢀ.0 (59/256)
1ꢀ.9 (ꢀ4/244)
0.01
P-value
P/AI, % (n/n)
Double-Ovsynch
GGPG
25.0 (2/8)
28.0 (14/50)
0.86
48.1 (ꢀ8/79)
46.7 (50/107)
0.85
5ꢀ.6 (59/110)
ꢀ0.2 (16/5ꢀ)
<0.01
59.ꢀ (ꢀ5/59)
61.8 (21/ꢀ4)
0.82
a,A
P-value
b,B
a,A
ab,A
Overall, % (n/n)
27.6 (16/58)
47.ꢀ (88/186)
46.0 (75/16ꢀ)
60.2 (56/9ꢀ)
a,b
Within the same row, means with different lowercase superscripts differ at P < 0.05.
Within the same row, means with different uppercase superscripts differ at P < 0.10.
A,B
1
Double-Ovsynch cows received the first GnRH injection of the Presynch portion of the Double-Ovsynch pro-
tocol at 5ꢀ ± ꢀ DIꢁ, followed by an injection of PGꢂ2α 7 d later and GnRH 72 h after PGꢂ2α, then began an
Ovsynch-56 protocol 7 d later. The GGPG cows received a GnRH injection at 6ꢀ ± ꢀ DIꢁ and then began
the Ovsynch-56 protocol 7 d later.
Journal of Dairy Science Vol. 97 No. 10, 2014

6312
CARVALHO ET AL.
tive. Presynchronization using a modified Ovsynch
protocol resulted in more cows with midrange P4 con-
centrations at G1 of the Ovsynch-56 protocol compared
with presynchronization using a single GnRH injection.
ꢁ
idrange P4 concentrations are an indication that
more DO cows were on d 7 of the estrous cycle (early
diestrus) when the Ovsynch-56 protocol was initiated
compared with GGPG cows. By contrast, more GGPG
cows were in the early and late stages of the estrous
cycle when the Ovsynch-56 protocol was initiated com-
pared with DO cows. Cows in the early stages of the
estrous cycle when an Ovsynch-56 protocol is initiated
have low P4 concentrations during development of the
ovulatory follicle. This low-P4 environment results in
overstimulation of the dominant follicle or oocyte by
exposure to a greater frequency of LH pulses (Revah
and Butler, 1996). This overstimulated oocyte is less
likely to produce a good quality embryo 7 d after AI
Figure 4. Distribution of cows based on progesterone concentra-
tions 4 d after timed AI (TAI) for cows presynchronized using a single
GnRH injection (GGPG; black bars) or a modified Ovsynch protocol
(
Double-Ovsynch; white bars) for first TAI. Double-Ovsynch cows re- (Rivera et al., 2011; Wiltbank et al., 2011). Even if a
ceived the first GnRH injection of the Presynch portion of the Double-
Ovsynch protocol at 5ꢀ ± ꢀ DIꢁ, followed by an injection of PGꢂ2α
good quality embryo develops, establishment of preg-
nancy is not guaranteed. In fact, cows with low P4 con-
protocol 7 d later, whereas GGPG cows received a GnRH injection at centrations during development of the ovulatory follicle
are more predisposed to prematurely develop pathways
leading to PGꢂ2α secretion by the endometrium causing
GnRH 56 h after PGꢂ2α, and were given a TAI 16 to 20 h after the short luteal phases (Cerri et al., 2011), thereby prevent-
7
d later and GnRH 72 h after PGꢂ2α, then began an Ovsynch-56
6
ꢀ ± ꢀ DIꢁ and then began the Ovsynch-56 protocol 7 d later. All
cows received a similar Ovsynch-56 protocol: at 70 ± ꢀ d cows received
a GnRH injection, followed by an injection of PGꢂ2α 7 d later and
last GnRH injection.
ing the establishment of pregnancy.
confinement-based dairies using a 7-d Ovsynch protocol
(
Souza et al., 2008; Herlihy et al., 2012), but not for cows
managed in grazing-based dairies using a 5-d Cosynch
protocol (Ribeiro et al., 2012). Despite having similar
P/AI, cows synchronized with a Double-Ovsynch pro-
tocol had fewer pregnancy losses compared with cows
synchronized with a Presynch-Ovsynch protocol (Ri-
beiro et al., 2012). In the present experiment, our objec-
tive was to compare synchronization of ovulation and
TAI using a Double-Ovsynch protocol with a simpler
protocol requiring fewer injections (a GGPG protocol)
at first TAI in high-producing dairy cows managed in a
confinement-based system. Based on data using hGPG
in resynchronized cows (Giordano et al., 2012b), our
hypotheses was that cows submitted for first TAI using
a GGPG protocol would have similar fertility to cows
receiving first TAI after a Double-Ovsynch protocol.
Contrary to our hypothesis, DO cows had more P/AI
at ꢀ2, 46, and 70 d after TAI compared with GGPG
cows. The underlying physiology that produces these
Figure 5. Distribution of cows based on progesterone concentra-
tions 11 d after timed AI (TAI) for cows presynchronized using a
single GnRH injection (GGPG; black bars) or a modified Ovsynch
protocol (Double-Ovsynch; white bars) for first TAI. Double-Ovsynch
dramatic differences in fertility appears to be a bet- cows received the first GnRH injection of the Presynch portion of
the Double-Ovsynch protocol at 5ꢀ ± ꢀ DIꢁ, followed by an injec-
ter synchrony of the estrous cycle for DO compared
with GGPG cows, as reflected by changes in circulating
tion of PGꢂ2α 7 d later and GnRH 72 h after PGꢂ2α, then began an
Ovsynch-56 protocol 7 d later, whereas GGPG cows received a GnRH
P4 at G1 and the PGꢂ2α injections of the Ovsynch-56 injection at 6ꢀ ± ꢀ DIꢁ and then began the Ovsynch-56 protocol 7
d later. All cows received a similar Ovsynch-56 protocol: at 70 ± ꢀ d
protocol.
cows received a GnRH injection, followed by an injection of PGꢂ2α
7
The distribution of cows based on P4 concentrations
d later and GnRH 56 h after PGꢂ2α, and were given a TAI 16 to 20 h
at initiation of the Ovsynch-56 protocol was illustra- after the last GnRH injection.
Journal of Dairy Science Vol. 97 No. 10, 2014

METHODS OF TIMED ARTIFICIAL INSEMINATION
6313
Cows that are in the late stages of the estrous cycle in the present study, and further studies are needed to
when an Ovsynch-56 protocol is initiated are less likely clarify reasons for the reduced P/AI in GGPG cows
to be properly synchronized, resulting in fewer P/AI with P4 concentration of 4 to 7 ng/mL at the time of
(
Vasconcelos et al., 1999). The reduced synchrony of the PGꢂ2α injection.
cows initiating an Ovsynch protocol during late diestrus Although circulating P4 after AI is essential for es-
might be partly attributed to a low ovulatory response tablishment and maintenance of pregnancy (Wiltbank
to the first GnRH injection of the protocol. Elevated et al., 2014), the relationship between post-AI P4 and
P4 concentrations at the time of the first GnRH injec- fertility is not conclusive. Supplementation with exoge-
tion decrease the amplitude of the GnRH-induced LH nous P4 during the early luteal phase after AI increased
surge (Giordano et al., 2012a). Cows in late diestrus the length of embryos (Carter et al., 2008; Clemente et
that failed to ovulate to the first GnRH injection of the al., 2009) possibly due to changes in gene expression in
protocol are more likely to have spontaneous luteolysis the uterus induced by P4 supplementation (Clemente
before the PGꢂ2α injection (Vasconcelos et al., 1999). et al., 2009). ꢁanipulative studies have used various
This leads to an LH surge before the last GnRH injec- methods to increase P4 after AI with only a marginal
tion and ovulation before TAI thereby decreasing the effect on P/AI (Wiltbank et al., 2014). ꢂor example,
chances of conception (Vasconcelos et al., 1999; ꢁoreira increasing P4 after AI by treating cows with human
et al., 2000).
chorionic gonadotropin 5 d after estrus to induce ac-
In addition, P4 concentrations at the PGꢂ2α injec- cessory CL increased P/AI by only ꢀ percentage points
tion further support the idea that more DO cows were compared with control cows (Nascimento et al., 201ꢀ).
in early diestrus when the Ovsynch-56 protocol was Although the relationship between post-AI P4 and P/
initiated. Independent of treatment, cows with low P4 AI is debatable (Wiltbank et al., 2014), a recent study
(
<1.0 ng/mL) at the PGꢂ2α injection had fewer P/ reported that fertility increased as P4 concentrations
AI compared with cows with high P4 (>1.0 ng/mL) increased after AI in cows synchronized using a Double-
concentrations, and more GGPG cows had low P4 con- Ovsynch protocol, but not for cows synchronized with
centrations at the PGꢂ2α injection compared with DO a Presynch-Ovsynch protocol (Herlihy et al., 2012). In
cows. Cows without a CL or with low P4 concentrations the present study, a reduction in P/AI in cows with
at the PGꢂ2α injection of the Ovsynch protocol have a lower (<0.ꢀ ng/mL) and higher (≥2.0 mg/mL) P4 at 4
greatly reduced P/AI compared with cows with high P4 d after AI was observed. The extremes in P4 (very low
at the PGꢂ2α injection (Bisinotto et al., 2010; Denicol or very high at 4 d after TAI) are likely indicative of
et al., 2012; Lopes et al., 201ꢀ). ꢂor example, during a asynchrony to the protocol rather than a direct effect
Resynch experiment (Giordano et al., 2012c), 25.6% of of P4 on embryonic growth. At 11 d after TAI, cows
cows had low P4 at the PGꢂ2α injection (111/4ꢀꢀ), and with low P4 (<2.0 ng/mL) had greatly reduced fertility
these cows had much lower fertility (8.1%) compared (11.6%) compared with cows with P4 exceeding 2 ng/
with cows with high P4 (ꢀ7.0%). In the present study, mL (5ꢀ%) probably indicating lack of synchrony to the
the reduction in P/AI for cows with low P4 concentra- protocol similar to observations in another study (Her-
tions at PGꢂ2α was also substantial (~20 percentage lihy et al., 2012). In contrast to another study (Herlihy
points), with most cows in this category (86%) being et al., 2012), when we removed these asynchronous
GGPG cows. The reduced fertility in this group of cows cows (1ꢀ.1% of cows) from the analysis, we observed
might be caused by a lack of synchrony due to prema- no effect on P/AI with increasing P4 at either 4 or
ture luteolysis leading to an LH surge and ovulation 11 d after TAI. Thus, our results indicate that post-
before TAI as discussed earlier.
AI P4 concentrations are useful for indicating lack of
An interesting observation from the present study synchrony to the timed AI protocol but do not support
was the reduced P/AI for GGPG (ꢀ0.2%) compared the idea that increasing P4 after AI improves P/AI at
with DO (5ꢀ.6%) cows with P4 concentrations from 4 least after submission of cows to a TAI protocol.
to 7 ng/mL at the PGꢂ2α injection. Reasons for this dif-
Interestingly, multiparous cows synchronized using
ference are not clear; however, they might be attributed a DO protocol had exceptional P/AI (52%) that was
to differences is synchronization of the follicular wave. similar to that of primiparous cows (55%). By contrast,
Double-Ovsynch cows were likely on d 7 on the estrous multiparous cows synchronized using a GGPG proto-
cycle and better responded to the first GnRH injection col had approximately 9 percentage points fewer P/AI
by ovulating a dominant follicle. By contrast, GGPG than primiparous cows; however, this difference did not
cows might have been in a later stage of the estrous reach statistical significance and may represent a Type
cycle at the first GnRH injection and therefore failed II error in the statistical analysis. Some studies using
to ovulate a dominant follicle leading to asynchrony of the Double-Ovsynch or GGPG protocols observed fewer
the follicular wave. We did not evaluate these endpoints P/AI for multiparous cows compared with primiparous
Journal of Dairy Science Vol. 97 No. 10, 2014

6314
CARVALHO ET AL.
cows (Souza et al., 2008; Giordano et al., 2012b; Lopes lington, WI) and the Dairy Cattle Center (ꢁadison,
et al., 201ꢀ), whereas others have reported no difference WI) for their help with data collection and cow handling
in P/AI between primiparous and multiparous cows during the course of this experiment. This research was
synchronized using a Double-Ovsynch protocol (Brus- supported by Hatch project WIS01171 to P. ꢁ. ꢂricke.
veen et al., 2009; Giordano et al., 2012c, 201ꢀ). Reasons
for the high P/AI in multiparous cows synchronized
using a Double-Ovsynch protocol in the present study
are not clear at this time; however, this difference con-
tributed to the increased P/AI for DO cows compared
with GGPG cows.
Heat stress causes a well-documented reduction in
fertility in lactating dairy cows (Rutigliano et al., 2008;
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CONCLUSIONS
Cows presynchronized for first TAI using a modified
Ovsynch protocol (i.e., a Double-Ovsynch protocol) had
more P/AI than cows presynchronized using a single
injection of GnRH 7 d before initiation of an Ovsynch
protocol (i.e., a GGPG protocol). This difference in
fertility was due to an increased synchrony during the
Ovsynch-56 protocol based on P4 concentrations col-
lected at G1 and the PGꢂ injections of the Ovsynch-56
2α
protocol. Thus, although a Double-Ovsynch protocol Edmonson, A. J., I. J. Lean, L. D. Weaver, T. ꢂarver, and G. Webster.
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ACKNOWLEDGMENTS
The authors thank the University of Wisconsin dairy
herd personnel at the Blaine Dairy Cattle Center (Ar-
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