Theriogenology 168 (2021) 75e82

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Theriogenology
journal homepage: www.theriojournal.com

Comparison of two intravaginal progesterone-releasing devices in

shortened-timed artificial insemination protocols in beef cattle
E.M. Zwiefelhofer a, K. Macmillan b, M. Gobikrushanth c, G.P. Adams a, S.X. Yang a, d,
M. Anzar a, d, M. Asai-Coakwell e, M.G. Colazo b, *
a
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, Canada
b
Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, T6G 2P5, Canada
c
Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, Canada
d
Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, S7N 0X2, Canada
e
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, S7N 5A8, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Commercially available intravaginal progesterone (P4) devices differ in shape, surface area and P4 load,
Received 15 October 2020 which may affect the resulting pregnancy per AI (P/AI) following timed-AI (TAI). The objective of this
Received in revised form study was to compare two intravaginal P4 devices on estrus rate, follicular dynamics and P/AI in beef
23 February 2021
cattle subjected to shortened-TAI protocols. In Expt. 1, nulliparous heifers were randomly assigned to a
Accepted 27 March 2021
P4-releasing intravaginal device (PRID-Delta, 1.55 g P4) or a controlled internal drug release (CIDR, 1.38 g
Available online 1 April 2021
P4) at the initiation of a J-synch protocol. Heifers that displayed estrus 72 h following device removal
were TAI, or if not in estrus given GnRH at 72 h and TAI at 90 h. In Expt. 2, nulliparous heifers and non-
Keywords:
PRID-Delta
suckling cows were randomly assigned to either PRID or CIDR groups and either 1 or 2 mg of estradiol
CIDR benzoate (EB) at initiation of a J-synch protocol. All cattle were TAI concurrent with GnRH 72 h after
Heifers device removal. In Expt. 3, nulliparous heifers and suckling cows were randomly assigned to either PRID
Cows or CIDR groups and initiated a 5-d Cosynch protocol, with TAI concurrent with GnRH 72 h following
P/AI device removal. In each experiment, cattle received estrus detection patches at device removal, which
Estrus rate were then scored from 0 to 3 based on color change between initial application and TAI; 0 ¼ unchanged,
1 ¼ 50% change, 2 ¼ >50% change, 3 ¼ missing. Estrus was defined to have occurred when the patch
was scored 2 or 3. Transrectal ultrasonography was used to determine cyclicity, diagnose pregnancy in all
experiments, and the size of the ovulatory follicle in Expt. 3. In Expt. 1, the estrus rate was greater (72.0%
vs. 61.0%; P ¼ 0.04) in the PRID compared to the CIDR group. In Expt. 2, a parity by EB dose interaction
(P ¼ 0.02) was attributed to an increased estrus rate (52.8% vs. 41.4%; P ¼ 0.05) in heifers given 1 vs.
2 mg EB. In Expt. 3, there was no difference in the ovulatory follicle diameter at device removal (P ¼ 0.22)
or TAI (P ¼ 0.28) between P4 groups. Treatment with a PRID tended (P ¼ 0.10) to increase the P/AI in cows
compared to a CIDR (73.5% vs. 61.0%). In all experiments combined, the overall P/AI tended to increase
(55.2% vs. 51.0%; P ¼ 0.08) and P/AI in cattle exhibiting estrus increased (64.4% vs. 59.7%; P ¼ 0.02) in
cattle given a PRID compared to those given a CIDR, respectively. In summary, the type of intravaginal P4
device affected estrus response and P/AI following TAI in beef cows.
© 2021 Elsevier Inc. All rights reserved.

1. Introduction benefit from AI by increased access to genetically superior bulls and

The use of artificial insemination (AI) in North America is lower
in beef than dairy cattle [1]; however, beef producers stand to
* Corresponding author. 410 Agriculture/Forestry Centre, University of Alberta,
Edmonton, Alberta, T6G 2P5, Canada.
E-mail address: colazo@ualberta.ca (M.G. Colazo).
a faster rate of genetic improvement in the herd. As well, AI may
increase the number of animals pregnant at the start of the
breeding season and require fewer clean-up bulls [1]. To increase
the adoption of AI in Canadian beef herds, it is important to develop
AI protocols that minimize handling, reduce or eliminate the need
for estrus detection and result in acceptable pregnancy per AI (P/AI)
[reviewed in 2].

https://doi.org/10.1016/j.theriogenology.2021.03.023
0093-691X/© 2021 Elsevier Inc. All rights reserved.
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al.
In synchronization protocols for timed-AI (TAI), exogenous
progesterone (P4) is used to suppress LH pulse frequency and delay
estrus and ovulation [2,3]. Removal of the exogenous P4 source
leads to a decrease in circulating P4 concentrations and stimulates
follicle maturation and ovulation [3]. First developed in the 1970s,
intravaginal P4 devices deliver sustained exogenous P4 [4]; the
controlled internal drug-releasing device (CIDR) and the P4
releasing intravaginal device (PRID-Delta) are the only devices
licensed for use in beef cattle in Canada. The PRID-Delta and CIDR
differ in shape (triangle vs. T-shape), P4 loads (1.55 vs. 1.38 g) and
surface areas (~155 vs. ~120 cm2 [4,5]). In a study comparing the
PRID-Delta and CIDR in dairy cattle, the PRID resulted in increased
circulating P4 concentrations and a tendency for increased P/AI [5].
Circulating P4 concentrations are negatively correlated with the
growth of follicles 8.5 mm as a result of a suppressive feedback
effect on LH pulse-frequency; that is, relatively low concentrations
of P4 are associated with overgrowth of the dominant follicle, and
vice versa [6]. Synchronization protocols utilizing P4 devices have
been shown to increase P/AI in beef cows compared to protocols
that did not use a P4 device [7]. Similarly, the amount of exogenous
P4 released during synchronization influenced fertility, as a twice-
used CIDR decreased P/AI compared to a new or once-used CIDR in
beef cattle subjected to a 7-d estradiol based protocol [8].
In addition to P4, TAI protocols combine several hormones to
produce tight synchrony of follicular wave emergence and ovula-
tion. In estradiol-based protocols, estradiol is given concurrent with
an intravaginal P4 device or ear implant to regress the dominant
follicle [9]. Follicle stimulating hormone (FSH) concentrations in-
crease 24e42 h after estradiol administration and stimulate the
synchronous emergence of a new follicular wave [9]. The effects of
estradiol benzoate (EB) on FSH is dose dependent [10], with greater
doses of EB delaying the pre-emergence increase in FSH. In
gonadotropin-releasing hormone (GnRH)-based protocols, admin-
istration of GnRH at P4 device insertion is intended to induce
ovulation of the current dominant follicle and initiate a new
follicular wave [11]. Recently, TAI protocols have been developed to
reduce the duration of P4 treatment and lengthen the period of
proestrus. A longer proestrus is associated with greater circulating
estradiol concentrations pre-ovulation and P4 concentrations post-
ovulation [12,13], in addition to increased P/AI [14,15]. In the J-
synch, an estradiol-based protocol, EB is given at the time of P4
device insertion, after 6 d prostaglandin F2a is administered at
device removal and 66e72 h later, TAI is performed concurrent
with GnRH administration [12]. In the 5-d Cosynch protocol, GnRH
is administered at the time of P4 device insertion, after 5 d the P4
device is removed, prostaglandin F2a given at device removal and
24 h later, and GnRH administered concurrent with TAI 66e72 h
after device removal [16]. However, the comparison between the
PRID and CIDR in beef cattle synchronized with shortened-TAI
protocols has not yet been reported. Therefore, there is a need to
determine the effect of the type of P4 device across the current
shortened-TAI protocols and parities on estrus response and P/AI.
Our rationale for the current study is that the PRID-Delta, with
an increased load of P4 and greater surface area, can be used in
shortened-TAI protocols to enhance fertility in beef cattle compared
to the CIDR. Three field experiments were done to compare the
effectiveness of two different intravaginal P4 devices for use in
different ovarian synchronization protocols for TAI in beef cattle.
The effect of a PRID-Delta vs. CIDR was compared in: 1) nulliparous
heifers synchronized for split-TAI using a J-synch protocol (Expt. 1),
2) nulliparous heifers and non-suckling primiparous cows given 1
vs. 2 mg of EB at the start of a J-synch protocol (Expt. 2), and 3)
nulliparous heifers and suckling cows synchronized with a 5-
d Cosynch protocol (Expt. 3).
76
Theriogenology 168 (2021) 75e82
2. Materials and methods
Expt. 1 was done in two commercial beef herds located in
Alberta, Canada from April to October 2019. Expt. 2 was done in one
commercial herd located in Alberta from June to August 2019. Expt.
3 was done at the University of Saskatchewan (Livestock and Forage
Centre of Excellence), Canada from June to August 2020. Procedures
were conducted in accordance with the guidelines of the Canadian
Council on Animal Care [17].
2.1. Experiment 1: Split-time artificial insemination using a J-synch
protocol
Nulliparous Angus and Simmental crossbred beef heifers
(n ¼ 470; 13e15 mo) were examined by transrectal ultrasonogra-
phy (Easi-scan, equipped with a multifrequency 4.5e8.5 MHz linear
transducer; BCF Technology LTD, Bellshill, Scotland, UK) to deter-
mine the presence of a corpus luteum (CL) to confirm cyclicity.
Heifers were assigned randomly to one of two groups and received
either 1) PRID-Delta (1.55 g of P4; Ceva Animal Health, Cambridge,
ON, Canada) or 2) CIDR (1.38 g of P4; Zoetis Canada Inc., Kirkland,
QC, Canada). Heifers were synchronized using the J-synch protocol,
consisting of 2 mg of EB i.m. (Summit Veterinary Pharmacy Inc., ON,
Canada) at the time of P4 device insertion, and 25 mg of dinoprost
tromethamine (Enzaprost T®, Ceva Animal Health) at the time of
device removal 6 d later. An estrus detection patch (Estrotect™,
Rockway Inc., Spring Valley, WI, USA) was applied cranial to the
tail-head at the time of P4 device removal and patches were scored
72 h later based on color change from initial application
(0 ¼ unchanged, 1 ¼ 50 color change, 2 ¼ >50 color change,
3 ¼ missing [18]). Heifers with a patch score of 2 or 3 were
considered in estrus and inseminated, while heifers with a score of
0 or 1 were given 100 mg gonadorelin i.m. (GnRH; Fertiline, Veto-
quinol N.A. Inc., Lavaltrie, QC, Canada) and inseminated at 90 h after
device removal. Heifers were inseminated by one technician using
commercially available, frozen-thawed semen from 1 of 6 sires (3
sires per farm), equally and randomly distributed between treat-
ments. Approximately 12 d after TAI, heifers were turned out to
pasture and exposed to 2- and 3-yr old Angus bulls (bull-to-heifer
ratio, approximately 1:50) for 32 d. Pregnancy diagnosis was done
by transrectal ultrasonography 52 d after TAI by the presence of a
viable embryo/fetus (heartbeat). Pregnancy from TAI was differ-
entiated from natural service based on embryo/fetus size [19].
2.2. Experiment 2: Dose of estradiol benzoate at initiation of a J-
Synch protocol
The experiment involved nulliparous Angus heifers (n ¼ 302;
15e17 mo) and non-suckling primiparous Angus cows (>6 mo
postpartum; n ¼ 88) with calves weaned at least 2 mo prior to
initiation of the study. Both heifers and cows were diagnosed non-
pregnant to a previous insemination. Using a 2  2 x 2 factorial
design, cattle (heifers vs. cows) at random stages of the estrous
cycle were assigned randomly to two different P4 groups (PRID-
Delta vs. CIDR) and given either 1 or 2 mg of EB i.m. at the time of P4
device insertion in a J-synch protocol. Estrus detection patches
were applied and scored, as described in Expt. 1. At 72 h after device
removal, cattle were given 100 mg GnRH concurrent with TAI per-
formed by one technician with frozen-thawed semen from one of
two sires which were equally and randomly distributed among
treatments. Pregnancy diagnosis was done by transrectal ultraso-
nography 40 d after TAI by the detection of an embryo with a
heartbeat.
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al.
2.3. Experiment 3: 5-d Cosynch protocol plus progesterone device
Angus and Hereford-cross nulliparous heifers (n ¼ 153;
14e17 mo) and suckling cows  42 days postpartum (n ¼ 208) were
enrolled in the 5-d Cosynch protocol, given 100 mg GnRH and
assigned randomly to either the PRID-Delta or CIDR group. The P4
device was removed 5 d later, concurrent with 500 mg of clopros-
tenol i.m. (Bioestrovet; Vetoquinol N.-A Inc.), which was repeated
24 h later. An estrus patch was applied and scored as described in
Expt. 1. At 72 h after device removal, all cattle were given 100 mg
GnRH concurrent with TAI performed by a single technician with
conventional frozen-thawed semen from 1 of 5 sires in heifers and
a single unit of pooled semen in cows, equally and randomly
distributed among treatments.
The ovaries were examined by transrectal ultrasonography
(MyLab 5 equipped with a 7.5 MHz linear array probe; Esaote N.A.,
Fishers, IN, USA) at P4 device insertion, device removal, and at TAI
to determine ovarian follicular dynamics, which were recorded on
an ovarian sketch sheet [20]. The diameter of the two largest fol-
licles were measured using built-in calipers on the ultrasound.
Ovulation to the initial GnRH treatment was confirmed at device
removal by the detection of a CL ipsilateral to the location of the
largest follicle (10 mm in diameter) detected at the time of initial
GnRH treatment. Following TAI, ovaries were examined by trans-
rectal ultrasonography daily for 2 d or until ovulation was detected,
as determined by the disappearance of a large follicle (10 mm in
diameter) from the previous examination. Pregnancy diagnosis was
done by transrectal ultrasonography 30 d after TAI as described in
Expt. 2.
2.4. Statistical analyses
Data were analyzed using SAS Enterprise Guide Version 9.4 (SAS
Institute Inc., Cary, NC, USA).
In Expt. 1, the proportion of heifers exhibiting estrus by AI, P/AI,
P/AI of heifers exhibiting estrus, and pregnancy to either AI or
natural service after first return to estrus were compared between
P4 device (PRID vs. CIDR) and AI timing (72 vs. 90 h) and their in-
teractions were analyzed as fixed effects using the GLIMMIX pro-
cedure. The effect of sire nested within location was used as a
random effect. The proportion of heifers exhibiting estrus by AI, P/
AI, P/AI of heifers exhibiting estrus, P/AI of heifers not exhibiting
estrus, and pregnancy to either AI or natural service at the end of
the breeding season were also compared between P4 device (PRID
vs. CIDR) and presence of CL (yes vs. no) and their interactions were
analyzed as fixed effects using the GLIMMIX procedure. In-
teractions were not significant and removed from the final model.
The model specifications included a binomial distribution and logit
function.
In Expt. 2, the proportion of heifers exhibiting estrus by TAI, P/
AI, and P/AI of heifers exhibiting estrus by TAI were compared
between P4 device (PRID vs. CIDR), EB dose (1 vs. 2 mg), parity (cow
vs. heifer) and their interactions were analyzed as fixed effects
using the GLIMMIX procedure. The final model included EB dose (1
vs. 2 mg), parity (cow vs. heifer), and their interactions. The effect of
sire was included as a random effect. The model specifications
included a binomial distribution and logit function.
In Expt. 3, the proportion of cattle that ovulated to GnRH at
device insertion, exhibited estrus by TAI, ovulated by TAI, ovulated
24 h after TAI, ovulated 48 h after TAI, ovulated overall, overall P/AI,
P/AI of cattle exhibiting estrus, and P/AI if ovulated to GnRH or did
not ovulate to GnRH at device insertion were compared between P4
device (PRID vs. CIDR), parity (cow vs. heifer), and their interactions
were analyzed as fixed effects using the GLIMMIX procedure. The
effect of sire was used as a random effect. The model specifications
77
Theriogenology 168 (2021) 75e82
included a binomial distribution and logit function. The ovulatory
follicle diameter on Day 5 and Day 8, and interval to ovulation were
compared between P4 device (PRID vs. CIDR), parity (cow vs.
heifer), and their interactions were analyzed using the MIXED
procedure.
The same endpoints in Expt. 3 were compared by further sub-
dividing the parity of the cows (Primiparous vs. Multiparous), P4
device (PRID vs. CIDR), and their interactions were analyzed using
either the GLIMMIX or MIXED procedure.
Data from the three experiments were combined and the pro-
portion of cattle exhibiting estrus by AI, P/AI, and P/AI of cattle
exhibiting estrus were compared between P4 device (PRID vs.
CIDR), parity (cow vs. heifer), and their interactions were analyzed
as fixed effects using the GLIMMIX procedure. The effect of sire
nested within location was used as a random effect. The P/AI was
compared among P4 device (PRID vs. CIDR), location (A, B, C, D), and
their interactions were analyzed as fixed effects using the GLIMMIX
procedure. The model specifications included a binomial distribu-
tion and logit function.
Statistical significance was defined as a P-value 0.05 and a
statistical tendency was defined as a P-value between 0.051 and
0.10.
3. Results
3.1. Experiment 1: Split-time artificial insemination using a J-synch
protocol
By 72 h after device removal, 243/470 [51.7%] of heifers were in
estrus and of the heifers that did not display estrus, an additional
70/227 [30.8%] were in estrus by 90 h. Overall, the P/AI for heifers
that were AI at 72 h was 143/243 [58.8%] and the P/AI for heifers AI
at 90 h was 83/227 [36.6%]. The P/AI of heifers that exhibited estrus
by AI was not different between 72 and 90 h (143/243 [58.8%] vs.
36/70 [51.4%]; P ¼ 0.30).
There were no interactions between P4 device and cyclicity for
any endpoint. A greater proportion of heifers exhibited estrus by AI
in the PRID compared to the CIDR group, respectively (172/239
[72.0%] vs. 141/231 [61.0%]; P ¼ 0.04; Table 1). In addition, there was
a tendency (P ¼ 0.08) for an increased P/AI in heifers that did not
exhibit estrus in the PRID compared to CIDR group, respectively
(24/67 [35.8%] vs. 23/90 [25.6%]; Table 1). A greater proportion of
cyclic heifers exhibited estrus compared to acyclic heifers (271/385
[70.4%] vs. 42/85 [49.4%]; P < 0.01). Overall P/AI (203/385 [52.7%]
vs. 23/85 [27.1%]; P <0.01), P/AI if estrus was exhibited (162/271
[59.8%] vs. 17/42 [40.5%]; P ¼ 0.02) or not exhibited by AI (41/114
[36.0%] vs. 6/43 [14.0%]; P ¼ 0.02), as well as the overall proportion
of heifers pregnant after AI and first bull breeding (306/385 [79.5%]
vs. 57/85 [67.1%]; P ¼ 0.02) was greater in cyclic compared to acyclic
heifers, respectively (Table 1).
3.2. Experiment 2: Dose of estradiol benzoate at initiation of a J-
Synch protocol
A total of 10 devices (6 PRID and 4 CIDR) were lost during
synchronization and data from those animals were removed from
further analyses. There was no effect of P4 device or interaction
between P4 device and parity or EB dose for any endpoint except
the proportion of cattle in estrus by AI, which tended (P ¼ 0.10) to
be lower in the PRID than CIDR group (77/188 [41.0%] vs. 102/192
[53.1%]). Overall P/AI (51/86 [59.3%] vs. 126/294 [42.9%]; P <0.01;
Table 2) and P/AI if exhibited estrus by AI (30/41 [73.2%] vs. 70/138
[50.7%]; P ¼ 0.01) was greater in cows than heifers, respectively.
There was no effect of EB dose on overall P/AI (94/185 [50.8%] vs.
83/195 [42.6%] for cattle administered 1 vs. 2 mg of EB; P ¼ 0.66).
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al. Theriogenology 168 (2021) 75e82

Table 1
Effect of cyclicity at the initiation of a J-synch protocol with either a PRID or a CIDR device on reproductive responses in nulliparous heifers (Expt. 1).

Variable PRID CIDR P-value

Cyclic Acyclic Cyclic Acyclic Device Cyclicity

n 203 36 182 49
Estrus rate 151/203 (74.4%) 21/36 (58.3%) 120/182 (65.9%) 21/49 (42.9%) 0.04 <0.001
P/AI (TAI) 107/203 (52.7%) 13/36 (36.1%) 96/182 (52.7%) 10/49 (20.4%) 0.14 <0.0001
P/AI (estrus) 87/151 (57.6%) 9/21 (42.9%) 75/120 (62.5%) 8/21 (38.1%) 0.93 0.02
P/AI (no estrus) 20/52 (38.5%) 4/15 (26.7%) 21/62 (33.9%) 2/28 (7.1%) 0.08 0.02
P/AI (TAI & NS) 157/203 (77.3%) 26/36 (72.2%) 149/182 (81.9%) 31/49 (63.3%) 0.83 0.02

Cyclicity was confirmed by the presence of a CL by transrectal ultrasonography prior to initiating the J-synch protocol.
Heifers were given 2 mg estradiol benzoate and either a progesterone-releasing intravaginal device (PRID-Delta) or a controlled internal drug release (CIDR) at initiation of a J-
synch protocol. Prostaglandin was given and the progesterone device was removed 6 days later. Heifers were inseminated at 72 h after device removal if estrus was displayed
(based on estrus detection patch scoring) or given 100 mg gonadorelin at 72 h and inseminated at 90 h after device removal. Bulls were turned out with heifers for natural
service (NS) approximately 12 d after AI and pregnancy diagnosis was done by transrectal ultrasonography to determine pregnancy per artificial insemination (P/AI) 52 d after
AI.
There was no interaction between P4 device and cyclicity for any endpoint.

There was a parity by EB dose interaction (P ¼ 0.02) for estrus rate
by AI, which was attributed to an increased estrus rate by AI in
heifers administered 1 mg compared to heifers administered 2 mg
of EB (75/142 [52.8%] vs. 63/152 [41.4%]; P <0.05; Table 2). There
was no effect of EB dose on P/AI in heifers nor on estrus rate by AI
and P/AI in cows.
3.3. Experiment 3: 5-d Cosynch protocol plus progesterone device
One heifer lost the PRID during synchronization, and one cow in
the PRID group, two heifers in the PRID group and one heifer in the
CIDR group became sick between TAI and pregnancy diagnosis;
their data were removed from further analyses. There were no P4
device by parity interactions for any endpoint (Table 3). A tendency
for an increased P/AI in cattle that exhibited estrus in the PRID
compared to the CIDR group was observed (93/124 [75.0%] vs. 85/
131 [64.9%]; P ¼ 0.09; Table 3). A greater proportion of cows ovu-
lated to the initial GnRH (115/208 [55.3%] vs. 54/152 [35.5%];
P < 0.001; Table 3) compared to heifers. However, a lesser pro-
portion of cows exhibited estrus by AI than heifers (136/208 [65.4%]
vs. 121/152 [79.6%]; P <0.01; Table 3). Ovulation by 24 h after TAI
was also determined in a lesser proportion of cows than heifers (95/
208 [45.7%] vs. 95/152 [62.5%]; P <0.01; Table 3), but a greater
proportion of cows ovulated by 48 h after TAI than heifers (88/208
[42.3%] vs. 38/152 [25%]; P <0.01; Table 3). Consequently, the in-
terval from TAI to ovulation was longer in cows compared to heifers
(32.0 ± 1.1 vs. 27.8 ± 1.2 h; P ¼ 0.01; Table 3). The diameter of the
ovulatory follicle was larger at P4 device removal (10.9 ± 0.1 vs.
10.5 ± 0.2 mm; P ¼ 0.03) and TAI (14.1 ± 0.1 vs. 13.3 ± 0.1 mm;
P <0.01) in cows than heifers, respectively (Table 3). In cattle that
did not ovulate to the initial GnRH, the ovulatory follicle at TAI
tended to be larger in cows compared to heifers (13.9 ± 0.2 vs.
13.3 ± 0.2 mm; P ¼ 0.08) and tended to be smaller in the PRID
compared to the CIDR group (13.3 ± 0.2 vs. 13.9 ± 0.2 mm; P ¼ 0.11)
There was no effect of parity or parity by P4 device interaction for P/
AI in cattle that did not ovulate to the initial GnRH; therefore, their
data were combined. The P/AI was numerically higher (P ¼ 0.18) in
the PRID vs. CIDR group (60/90 [66.7%] vs. 58/100 [58.0%]) in cattle
that did not ovulate to the initial GnRH.
When only data from cows were analyzed, P/AI tended
(P ¼ 0.10) to be increased in the PRID compared to the CIDR group
(75/102 [73.5%] vs. 64/105 [61.0%]; Table 4). In addition, there was a
greater overall P/AI (106/148 [71.6%] vs. 33/59 [55.9%], P ¼ 0.04) and
increased P/AI in cows that exhibited estrus by TAI (74/94 [78.2%]
vs. 24/42 [57.1%]; P <0.01) for multiparous compared to primipa-
rous cows, respectively (Table 4).
3.4. Experiments combined
There were no interactions between P4 device and location.
Overall, P/AI tended to be greater in cattle receiving a PRID than
cattle receiving a CIDR (334/605 [55.2%] vs. 308/604 [51.0%];
P ¼ 0.08; Fig. 1) and was greater in cows than heifers (190/293
[64.8%] vs. 452/916 [49.3%]; P <0.01; Fig. 1). Cattle that exhibited
estrus by TAI had increased P/AI (215/334 [64.4%] vs. 206/345
[59.7%]; P ¼ 0.02) in the PRID compared to the CIDR group,
respectively. In addition, cows exhibiting estrus by TAI had greater
P/AI compared to heifers exhibiting estrus (128/177 [72.3%] vs. 293/
502 [58.4%]; P <0.01). There was also a P4 device by parity inter-
action (P ¼ 0.02), in which cows given a PRID and exhibiting estrus
by TAI (68/83 [81.9%]) had increased P/AI compared to both PRID
and CIDR heifers exhibiting estrus (147/251 [58.6%] and 146/251

Table 2
Effect of parity and dose of estradiol benzoate given at the initiation of a J-synch protocol on estrus expression and pregnancy per AI (P/AI) in nulliparous heifers and non-
suckling cows (Expt. 2).

Variable Cows Heifers P-value

1 mg EB 2 mg EB 1 mg EB 2 mg EB Parity EB dose

n 44 44 146 156
Estrus by AI1 17/43 (39.5%)ab 24/43 (55.8%)ab 75/142 (52.8%)a 63/152 (41.4%)b 0.95 0.67
P/AI overall 24/43 (55.8%) 27/43 (62.8%) 70/142 (49.3%) 56/152 (36.8%) <0.01 0.66
P/AI estrus by AI 11/17 (64.7%) 19/24 (79.2%) 45/75 (60%) 25/63 (39.7%) 0.01 0.64

Cows and heifers were administered either 1 or 2 mg of estradiol benzoate intramuscularly at the insertion of an intravaginal progesterone device (PRID-Delta or CIDR).
Prostaglandin was given and the progesterone device was removed 6 days later. Timed artificial insemination (TAI) was done 72 h later and pregnancy diagnosis was done by
transrectal ultrasonography 40 days after TAI.
There was no effect of progesterone device for any endpoint.
1
Parity by EB dose interaction (P ¼ 0.02).
ab
Within a row, values without a common superscript differ (P < 0.05).

78
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al. Theriogenology 168 (2021) 75e82

Table 3
Follicle diameter, ovulation and pregnancy per AI (P/AI) in beef cattle synchronized with a 5-d Cosynch protocol plus either a PRID or CIDR device and timed artificial
insemination (TAI) at 72 h after device removal in nulliparous heifers and suckling cows (Expt. 3).

Variable PRID CIDR P-value

Cows Heifers Cows Heifers Device Parity

n 103 76 105 76 e e
Ovulated to initial GnRH 62/103 (60.2%) 26/76 (34.2%) 53/105 (50.5%) 28/76 (36.8%) 0.53 <0.001
Ovulatory follicle diameter (mm) at:
Device removal 10.8 ± 0.2 10.4 ± 0.2 11.1 ± 0.2 10.6 ± 0.2 0.22 0.03
TAI 14.0 ± 0.2 13.2 ± 0.2 14.3 ± 0.2 13.4 ± 0.2 0.28 <0.0001
Estrus by AI 64/103 (62.1%) 61/76 (80.2%) 72/105 (68.6%) 60/76 (78.9%) 0.68 <0.01
Ovulation detected at TAI 12/103 (11.7%) 7/76 (9.2%) 10/105 (9.5%) 8/76 (10.5%) 0.79 0.96
Ovulation detected 24 h after TAI 42/103 (40.7%) 48/76 (63.2%) 53/105 (50.5%) 47/76 (61.8%) 0.20 <0.001
Ovulation detected 48 h after TAI 49/103 (47.6%) 20/76 (26.3%) 39/105 (37.1%) 18/76 (23.7%) 0.25 <0.001
Cumulative ovulation 103/103 (100%) 74/76 (97.4%) 102/105 (97.1%) 74/76 (97.4%) 0.97 0.97
Interval to ovulation from TAI (h) 32.5 ± 1.6 28.3 ± 1.7 31.3 ± 1.5 27.2 ± 1.6 0.46 0.01
P/AI following TAI 75/102 (73.5%) 50/74 (67.6%) 64/105 (61.0%) 50/75 (66.7%) 0.21 0.81
P/AI estrus by TAI 52/64 (81.3%) 41/60 (68.3%) 46/72 (63.9%) 39/59 (66.1%) 0.09 0.43
P/AI if ovulated to GnRH at initiation of synchronization 48/62 (77.4%) 17/26 (65.4%) 36/53 (67.9%) 20/28 (71.4%) 0.98 0.59

Cattle were given 100 mg gonadorelin (GnRH) at the insertion of either a progesterone-releasing intravaginal device (PRID-Delta) or a controlled internal drug release (CIDR)
for 5 days. At the time of device removal an estrus detection patch was applied and 500 mg cloprostenol given to induce luteolysis,which was repeated 24 h later. Cattle were
timed artificially inseminated (TAI) concurrent with GnRH 72 h after device removal. The estrus detection patch was scored from 0 to 3, based on color change from initial
application (0 ¼ unchanged, 1 ¼ 50 color change, 2 ¼ >50 color change, 3 ¼ missing). Estrus was defined as a score 2. Ovulatory follicle diameter, ovulation, and pregnancy
per artificial insemination (P/AI) were determined by transrectal ultrasonography.
There was no interaction between P4 device and parity for any endpoint.

[58.2%]). Cows not exhibiting estrus by TAI had increased P/AI
compared to heifers not exhibiting estrus (62/116 [53.4%] vs. 159/
414 [38.4%]; P ¼ 0.02).
4. Discussion
Timed-AI protocols that maximize pregnancy success and
minimize labor requirement may lead to more Canadian beef
producers adopting these protocols for genetic improvement of
their herds. Until recently, the CIDR was the only commercially
available P4 device for estrus synchronization in beef and dairy
cattle in Canada. The recently approved PRID-Delta can now also be
incorporated in shortened TAI protocols to improve pregnancy
success.
Across the experiments in the current study, the significant
differences between the PRID and CIDR were an increase in estrus
rate in heifers and a higher P/AI among cows that exhibited estrus.
There were also several tendencies for difference in P/AI between
the PRID and CIDR, in which the PRID tended to improve overall P/
AI in cattle exhibiting estrus and in heifers not exhibiting estrus. In
this regard, van Werzen et al. [5] also reported a similar tendency
for increased P/AI in lactating dairy cows treated with a PRID-Delta
compared to those treated with a CIDR, but no interactions with
parity or cyclicity. As circulating drug concentrations are correlated
to intravaginal device surface area, it is speculated that the PRID,
which contains 29% more surface area than the CIDR, would result
in increased circulating P4 concentrations. In ovariectomized dairy
cows, the PRID-Delta resulted in increased circulating P4 concen-
trations compared to the CIDR [5], which may be due to the dif-
ference in P4 concentrations and/or surface area of the devices.
Using suckling beef cows in Expt. 3, the difference in P/AI between
the two P4 devices was more noticeable than in Expt. 1 and 2,

Table 4
Follicular and pregnancy data in cows synchronized with a 5-d Cosynch protocol plus either a PRID or CIDR device (Expt. 3) and timed artificial insemination (TAI) at 72 h after
device removal in suckling cows.

Variable PRID CIDR P-value

Multiparous Primiparous Multiparous Primiparous Device Parity

n 76 27 73 32
Ovulated to initial GnRH 43/76 (56.6%) 19/27 (70.4%) 36/73 (49.3%) 17/32 (53.1%) 0.11 0.24
Ovulatory follicle diameter (mm) at: 10.9 ± 0.2 10.4 ± 0.4 11.0 ± 0.2 11.1 ± 0.3 0.46 0.19
Device removal
TAI 14.1 ± 0.2 13.7 ± 0.4 14.2 ± 0.2 14.5 ± 0.4 0.13 0.86
Estrus by TAI 46/76 (60.5%) 18/27 (66.7%) 48/73 (65.8%) 24/32 (75%) 0.35 0.29
Ovulation detected at TAI 9/76 (11.8%) 3/27 (11.1%) 6/73 (8.2%) 3/32 (9.4%) 0.56 0.94
Ovulation detected 24 h after TAI 30/76 (39.5%) 12/27 (44.4%) 35/73 (47.9%) 18/32 (56.3%) 0.19 0.39
Ovulation detected 48 h after TAI 37/76 (48.7%) 12/27 (44.4%) 32/73 (43.8%) 8/32 (25%) 0.33 0.37
Cumulative ovulation 76/76 (100%) 27/27 (100%) 73/73 (100%) 29/32 (90.6%) 0.97 0.97
Interval to ovulation from TAI (h) 32.8 ± 1.9 32.0 ± 3.1 32.5 ± 1.8 28.1 ± 2.7 0.40 0.29
P/AI following TAI 58/75 (77.3%) 17/27 (63.0%) 48/73 (65.8%) 16/32 (50.0%) 0.10 0.04
P/AI estrus by TAI 41/46 (89.1%) 11/18 (61.1%) 33/48 (68.8%) 13/24 (54.2%) 0.12 <0.01
P/AI if ovulated to initial GnRH 37/43 (86.0%) 11/19 (57.9%) 26/36 (72.2%) 10/17 (58.8%) 0.32 0.02

Cows were given 100 mg gonadorelin (GnRH) at the insertion of either a progesterone-releasing intravaginal device-Delta (PRID) or a controlled internal drug release (CIDR) for
5 days. At the time of device removal and 24 h later, cattle were given 500 mg cloprostenol to induce luteolysis and an estrus detection patch was applied. Cows were timed
artificially inseminated (TAI) concurrent with GnRH 72 h after device removal. The estrus detection patch was scored from 0 to 3, based on color change from initial application
(0 ¼ unchanged, 1 ¼ 50 color change, 2 ¼ >50 color change, 3 ¼ missing). Estrus was defined as a score 2. Ovulatory follicle diameter, ovulation, and pregnancy per artificial
insemination (P/AI) were monitored by transrectal ultrasonography.
There was no interaction between P4 device and parity for any endpoint.

79
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al.
Fig. 1. Combined pregnancy per AI (P/AI) in cows and heifers given either a
progesterone-releasing intravaginal device (PRID-Delta) or a controlled internal drug
release (CIDR) during synchronization for timed artificial insemination from all ex-
periments. The boxed number in each column represents the total number of animals
per treatment or parity.
There was no interaction between progesterone device and parity (P ¼ 0.30).
which involved heifers and non-suckling cows. Therefore, it may be
hypothesized that either a CIDR does not allow threshold concen-
trations of P4 to be achieved in suckling beef cows or increased P4
metabolism exists in suckling beef cows in a similar manner as
shown in high producing lactating dairy cows [21]. The inclusion of
one CIDR in lactating dairy cows has been shown to increase
circulating P4 concentrations by only 0.8 ng/mL [22], whereas
serum P4 concentrations 2 ng/mL tended to increase P/AI in
anestrus dairy cows given two CIDR devices during follicular
growth [23]. Moreover, our group has shown that a twice-used
CIDR decreased P/AI compared to a new (containing 1.9 g of P4)
or a once-used CIDR following TAI in beef cattle subjected to a 7-
d estradiol-based protocol [8]. The objective of the current study
was to compare P/AI between the P4 devices in field trials and not
to evaluate P4 concentrations, which has already been reported [5].
It is speculated that the increased P/AI in the PRID vs. CIDR may be
attributed to the PRID possibly providing more P4 release than the
CIDR in beef cattle.
The influence of increased P4 concentrations during follicular
growth on pregnancy success in a shortened-TAI prototol has not
been fully elucidated. Progesterone inversely regulates LH pulse
frequency which impacts the growth of follicles 8.5 mm in
diameter (reviewed in Ref. [6]) resulting in smaller ovulatory fol-
licles [24], but increased P4 also promotes oocyte quality (reviewed
in Ref. [25]). In the current study, the average diameter of the
ovulatory follicle at the time of device removal was numerically
smaller in cows in the PRID vs. CIDR group. High P4 concentrations
during follicular growth has been also shown to increase intra-
follicular IGF-1 [26] and benefit early embryo development post
ovulation in beef and dairy cattle [27,28]. Conversely, low P4 con-
centrations during follicular growth increased circulating estradiol
[29], shortened the interval to estrus and ovulation due to hastened
luteolysis [30], which may result in lower P/AI [31]. Following EB
administration, follicular wave emergence of follicles ~4 mm in
diameter is expected to occur 3e4 d later [10]. Assuming that the
granulosa cells on the dominant follicle acquire LH responsiveness
at 8.5 mm in diameter (i.e., at the time of follicle deviation)
approximately 3 d after wave emergence [6], in the J-synch pro-
tocol, P4 inversely regulates dominant follicle growth for a short
time before the device is removed and prostaglandin F2a is given.
Similarly, in the 5-d Cosynch protocol, if ovulation of the dominant
follicle occurs, a new follicular wave emerges on average 2 d after
GnRH administration [32], thus, the dominant follicle becomes LH
80
Theriogenology 168 (2021) 75e82
responsive at the time of device removal and prostaglandin F2a
administration. In Expt. 3, cattle that did not ovulate to the initial
GnRH of Cosynch had a tendency for a decreased ovulatory follicle
diameter and a numerically increased P/AI in the PRID compared to
CIDR group. This difference seems to support the negative effect of
P4 on LH frequency and follicle size, resulting in an increased P/AI.
However, the role of P4 regulating LH frequency before follicle
deviation cannot be ruled out as the theca interna cells expressed
LH-receptor mRNA beginning at ~0.5 mm in diameter that
increased linearly with follicular size [33]. Recently, it was shown
that increased P4 resulted in an undersized (<7 mm diameter)
subordinate follicle at the time of follicle deviation, providing evi-
dence that P4 may regulate the size of follicles pre-deviation [34].
However, the effect of P4 (and hence LH frequency) on follicles
<8.5 mm has not been fully elucidated and warrants further
research. In addition, P4 is essential for the preparation of an
optimal uterine environment before breeding, which is of partic-
ular importance in postpartum suckling cows and prepubertal
heifers. In Expt. 1, there was a numerical increase in P/AI in acyclic
heifers given a PRID compared to a CIDR, thus, future studies are
also warranted using beef cattle with low circulating P4 at the
beginning of synchronization for TAI. Overall, exposure to high P4
concentrations during the growth of the dominant follicle and
before the administration of prostaglandin F2a appear to improve
fertility and may have resulted in greater P/AI for cattle given a PRID
in the current study.
In Expt. 2, there was no effect of EB dose on P/AI but there was an
effect on estrus rate in heifers. The decreased estrus response by TAI
in heifers given 2 mg compared to 1 mg of EB is most likely
explained by the metabolism of EB. In suckling beef cows given 1
and 2 mg of EB, the metabolism of EB led to peak FSH concentra-
tions at 53 and 81 h after treatment, respectively [10]. Using heifers
in the present study, it is assumed maximum circulating EB con-
centrations were higher and that the time to reach nadir EB levels
(and peak FSH) were longer than in cows, delaying the onset of new
follicular wave emergence. It is hypothesized that the lower estrus
response in heifers given 2 mg EB was due to delayed wave
emergence which resulted in a smaller ovulatory follicle diameter
than the 1 mg EB group by TAI. However, this remains unknown as
ovulatory follicle diameter at TAI or blood estradiol concentrations
were not determined to confirm our hypothesis. It is also important
to note that the nature of the experimental design for this study
resulted in reduced statistical power. In regard to P/AI, results from
the current study are similar to previous work in both dairy and
beef cows, which reported no difference in P/AI when larger doses
of EB were used in a TAI protocol [35,36]. Overall, results suggest
that administering 2 mg of EB in a J-synch protocol is unnecessary
and may reduce estrus response in heifers.
In addition to experimental treatments in the current study,
other factors also played a role in reproductive outcomes. For
heifers in Expt. 1, cyclicity resulted in improved estrus response and
P/AI. Previous studies have also reported an increase in estrus
response in cyclic beef heifers [18,37] and an increase in P/AI [38]
compared to acyclic heifers. This could be due to estrus near the
time of AI resulting in increased P/AI [18,37]. Although reproductive
protocols between experiments cannot be compared, the split-
timed AI in Expt. 1 allowed for an additional 15% of heifers to
display estrus before AI and total estrus response (67 vs. 47%) and P/
AI (48 vs. 43%) was greater than for heifers in Expt. 2. However,
cyclic heifers in Expt. 1 had increased P/AI regardless of expression
of estrus at TAI. In Expt. 3, parity also influenced fertility. Overall, P/
AI was greater in cows compared with heifers and in multiparous
cows compared with primiparous. Previous studies have also re-
ported a reduced P/AI in primiparous compared to multiparous
beef cows [39], which may be due to a lower energy balance post-
E.M. Zwiefelhofer, K. Macmillan, M. Gobikrushanth et al.
calving as a larger amount of energy and nutrients is still required
for growth [40]. It is interesting to note that for cows submitted to
the 5-d Cosynch, there was a greater ovulatory response to the
initial GnRH, but fewer cows exhibited estrus by AI and ovulated
within 24 after AI compared to heifers. Previous work has shown
that a low proportion of heifers ovulate to the initial GnRH [15] and
the current results indicate that ovulation response may delay
luteal regression, estrus and ovulation after TAI. This may have
resulted in heifers and cows having similar P/AI in Expt. 3, in which
absence of estrus at TAI [18,37] and ovulation occurring greater
than 24 h after AI [41] was associated with reduced P/AI. In Expt. 3,
there was a weak tendency for an increased proportion of cows that
ovulated following the initial GnRH in the PRID compared to CIDR
group. As the pituitary response to GnRH administration is within
minutes [42], this finding is hypothesized to be mainly due to the
size and stage of development of the dominant follicle [43] and the
endogenous P4 concentrations at the time of GnRH administration
[44] rather than the speculated increase in P4 concentrations
generated by the P4 device itself. It is important to consider
cyclicity, estrus response and parity when evaluating reproductive
protocols.
5. Conclusion
In summary, treatment with a PRID tended to increase the P/AI
when compared to the CIDR in beef cattle. In addition, using a PRID
during a J-synch protocol increased the estrus rate by AI compared
to a CIDR in nulliparous heifers. There was no effect of EB dose
concentration on P/AI, although heifers given 1 mg of EB at the
initiation of a J-synch protocol had greater estrus rate by TAI
compared to 2 mg of EB. Finally, there was no effect of P4 device
during synchronization on follicular dynamics in nulliparous
heifers and suckling cows, but treatment with a PRID increased P/AI
in cows exhibiting estrus by TAI. Pregnancy per AI was also
increased in cyclic compared with acyclic heifers and in multipa-
rous compared to primiparous cows. Future studies are warranted
to further investigate the effect of an intravaginal device with an
increased P4 load during synchronization in both acyclic heifers
and suckling cows subjected to shortened-TAI protocols.
CRediT authorship contribution statement
E.M. Zwiefelhofer: Formal analysis, Methodology, Investigation,
Writing e original draft, Visualization. K. Macmillan: Methodology,
Investigation, Writing e original draft, Visualization. M. Gobik-
rushanth: Formal analysis, Investigation, Writing e review &
editing, Visualization. G.P. Adams: Methodology, Writing e review
& editing, Supervision, Funding acquisition. S.X. Yang: Investiga-
tion, Writing e review & editing. M. Anzar: Writing e review &
editing, Supervision. M. Asai-Coakwell: Investigation, Writing e
review & editing. M.G. Colazo: Conceptualization, Methodology,
Investigation, Writing e review & editing, Supervision, Funding
acquisition.
Acknowledgements
Research was supported by Livestock Systems Section, Alberta
Agriculture and Forestry, Saskatchewan Agriculture Development
Fund, and Agriculture and Agri-Food Canada. Authors thank Ceva
Animal Health (Cambridge, ON, Canada) and Rockway Inc. (Spring
Valley, WI, USA) for their in-kind support and the Crow family
(Thorsby, AB, Canada), Holt Hutterite colony (Kinsella, AB, Canada)
and Parkland Cattle Co. (Spruce Grove, AB, Canada) for their
cooperation during the study. Authors also thank Dr. Graeme
Boender (The Farm Animal Hospital, Leduc, AB, Canada), Dr. Laura
81
Theriogenology 168 (2021) 75e82
Macrae (Agri-Vet Alberta, Westlock, AB, Canada), Miranda Zwie-
felhofer, Jessie Hellquist, Hailey Bolen, Crystal Ketel and the staff at
the Livestock and Forage Centre of Excellence for technical assis-
tance during the study.
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