Introduction

In sheep, estrus synchronization protocols increase reproductive efficiency through hormone administration¹. The aim is to modify the estrus cycle such that lambs are produced in uniform batches, thus reducing the costs of reproductive management. These protocols are traditionally implemented for 12 to 14 d using controlled internal drug release (CIDR) intravaginal devices (such as inserts and sponges) containing natural progesterone². However, prolonged use of progestogens obstruct sperm transport³ and modifies follicular dynamics⁴.

Progestogens’ negative effects can be reduced through short synchronization protocols², that is, application of CIDR devices for five to seven days. In addition, prostaglandin F₂ alpha (PGF₂α) is administered to control luteal function, as is equine chorionic gonadotropin (eCG) to stimulate follicular development and ovulation by increasing both the number of follicles and the estrogen production rate of the stimulated follicles⁴. This produces a follicle with a greater capacity for fertilization during the reproductive season and seasonal anestrus⁵.

The physical characteristics of CIDR inserts allow them to be washed and reused without reducing reproductive variables⁶. When short synchronization protocols of five^6,7 and seven days^8,9,10 are used in sheep, inserts retain a considerable amount of biologically-available progesterone (P₄) after withdrawal^3,11. Reuse of CIDR inserts remains controversial. Some authors report reduced estrus and pregnancy synchronization rates with reused inserts^6,11, and others³ have found higher fertility and fecundity rates with new CIDR inserts than with those used a second and third time. It has also been suggested that, in inserts used for up to six times, reproductive variables begin to decrease beginning with the second reuse³.

In contrast, there are reports that insert reuse has no effect on reproductive variables. One study comparing second- and third-use inserts to new inserts in six-day treatments found no differences in estrus synchronization rate and ovulatory response, although blood progesterone concentrations and pregnancy rates were lower in ewes treated with reused inserts¹¹. Another study found that when used up to three times in six-day protocols, inserts produced similar pregnancy rates with both new and reused devices¹².

Short synchronization protocols theoretically allow for repeated CIDR insert use. This implies a decrease in production costs, a reduction in ewe exposure to progesterone (P₄) concentrations to half that recommended by the manufacturer, and the consequent possibility of using CIDR inserts with lower hormone content and without affecting reproductive performance. When CIDR inserts are used in two 12-d protocols, the main reproductive variables do not differ between the first and second uses because the inserts contain sufficient P₄ to maintain luteotropic function and synchronize estrus in sheep¹³. Based on this result, if inserts are used up to four times in six-day protocols, reproductive variables (ewes in estrus, pregnancy rate, fertility rate) can be expected to remain at the same or similar values to those in new CIDR inserts.

The objective of the present study was to test the effect of new and second-, third- and fourth-use CIDR inserts in six-day estrus synchronization protocols on reproductive performance and progesterone concentrations in multiparous ewes.

Material and methods

The study was done during the sheep reproductive season (June - December, 2020) at Montecillo, in the municipality of Texcoco, State of Mexico, Mexico (19°27’18” N, 98°54’26” W; 2220 m asl). Study area climate is subhumid temperate with summer rains¹⁴.

Animals, feeding and treatments

Experimental animals were 172 multiparous female sheep (Katahdin × Dorset) with 59 kg average weight and a body condition of 3 (1-to-5 scale). They were housed in pens, fed a diet of oat hay with grains, alfalfa hay and 300 g commercial breeding sheep feed (PURINA^®; 14% CP), and provided free access to water. Prior to beginning the study, the animals were subjected to prophylactic management including a multivitamin (Endovet^®; 0.5 ml/ 50 kg L.W., i.m.) and a bacterin-toxoid (Bobact^® 8; 2.5 ml subcutaneously), the absence of pregnancy was confirmed by ultrasound. The animals were managed in compliance with federal regulations for the use and care of research animals^15,16.

The animals were randomly distributed into four experimental groups (n=43 sheep/group); each group was administered a progesterone hormone treatment via intravaginal devices (Eazi Breed™ CIDR^® 330 sheep & goat insert, Zoetis), 200 IU equine chorionic gonadotropin (eCG) (NOVORMON 5000^®, Zoetis; i.m.) and 125 µg prostaglandin (Celosil^®, M.S.D). The four treatments were CIDR1 (control group, new devices), CIDR2 (second-use devices), CIDR3 (third-use devices) and CIDR4 (fourth-use devices). All reused CIDR inserts were thoroughly washed with purified water, dried, and stored under refrigeration for up to 24 h prior to reinsertion. In the estrus synchronization protocol, the devices were inserted on day -7 (d-7) and removed on day -1 (d-1); eCG and prostaglandin were applied intramuscularly at removal. Estrus onset was considered to occur on day 0 (d0) (Figure 1).

Figure 1 Estrus synchronization protocol and blood sample timeline 

Results

Average ewes in estrus rate was 97 % and did not differ (P>0.05) between treatments. Average estrus onset was 36 ± 11 h. The ewes in CIDR2 began estrus in less time and in a shorter interval than those in CIDR1, while CIDR3 and CIDR4 did not differ (P>0.05) from CIDR1 and CIDR2. No differences were identified in the variables of pregnancy rate (81 %), fertility rate (80 %), single parturitions (70 %) and double parturitions (30 %) (Table 1).

Most (94 %) of the animals exhibited estrus 54 h after device removal, although in treatment CIDR2 this did not occur until 66 h and in CIDR3 until 72 h (Figure 2).

CIDR1= new devices; CIDR2 = second-use; CIDR3 = third-use; CIDR4 = fourth-use. * ± standard deviation.

^ab Different superscript letters in the same set of bars indicate significant difference (P≤0.05).

Figure 2 Percentage of ewes in estrus 

Mean progesterone (P₄) concentrations did not differ (P>0.05) between treatments on the day (d-7) the devices were inserted (CIDR1 = 2.12 ± 0.72, CIDR2 = 1.46 ± 60, CIDR3 = 1.54 ± 0.49, CIDR4 = 2.78 ± 0.52 ng/ml). While inserted, the highest P₄ concentrations in all treatments were observed three days after insertion (d-4). On this day, the highest (P<0.05) concentration was in CIDR1 (6.78 ± 1.23 ng/ml), followed by CIDR2 (4.90 ± 1.36) and CIDR 3 (4.97 ± 1.37 ng/ml), which did not differ, and CIDR4 (2.78 ± 1.56 ng/ml), the lowest (P<0.05) (Figure 3).

^ab Different superscript letters in the same day indicate significant difference (P≤0.05).

Figure 3 Serum progesterone (P₄) concentrations in ewes after application of CIDR devices (new, second-use, third-use and fourth-use) 

Immediately after CIDR device removal (d-1), P₄ concentrations in the CIDR1 (4.39 ± 0.98 ng/ml), CIDR2 (4.75 ± 0.79 ng/ml) and CIDR3 (4.86 ±0.75 ng/ml) treatments were higher (P≤0.05) than in CIDR4 (2.48 ± 1.52 ng/ml). However, 24 h later (d0), P₄ concentrations had decreased to levels that did not differ (P>0.05) between treatments (CDIR1= 1.38 ± 0.26, CDIR2= 1.07 ± 0.39, CDIR3= 1.38 ± 0.15, CDIR4= 0.74 ± 0.23).

By day d2 (72 h after device withdrawal), 97 % of the animals exhibited estrus and average P₄ was <1 ng/ml in CIDR1 (0.93 ± 0.04 ng/ml), CIDR2 (0.62 ± 0.06 ng/ml) and CIDR4 (0.71 ± 0.6 ng/ml), and only slightly higher in CIDR3 (1.01 ± 0.14 ng/ml). The treatments did not differ (P>0.05).

At d8, all four treatments exhibited P₄ concentrations >1 ng/ml with no differences between them (P>0.05) (CIDR1= 4.64 ± 1.24, CIDR2= 5.09 ± 1.05, CIDR3= 5.11 ± 1.85 and CIDR4= 3.86 ± 1.11 ng/ml. By d15, which corresponds to endometrium adhesion (12 to 13 days pregnancy), concentrations were highest (P<0.05) in CIDR2 (7.56 ± 0.85 ng/ml) and CIDR3 (7.95 ± 1.0 ng/ml) and lowest (P<0.05) in CIDR1 (5.43 ± 0.97 ng/ml) and CIDR4 (5.52 ± 1.04).

Discussion

The 97 % ewes in estrus observed in the present results is similar to that reported in a study using five-day protocols (93 % in second-use devices and 100 % in third-use devices)⁶. The ewes in estrus results observed here suggest that CIDR devices used up to four times in six-day protocols are as effective as new devices in synchronizing estrus and ovulation during the reproductive season^6,11. Indeed, this ewes in estrus rate is similar to those reported for conventional 12-d treatments¹³.

The mean estrus onset results were like those reported for seven-day protocols with new CIDR devices associated with 5 mg dinoprost and 400 IU eCG (33.8 ± 4.0 h)². They were also similar to the 35 ± 0.7 h estrus onset reported at 7 d with devices with 11 d previous use and associated with 300 IU eCG and 6.7 mg dinoprost, and for which ewes in estrus was highest between 36 and 41.9 h¹⁰. The low ewes in estrus rates after 54 h in CDIR2 (minimum 3 %) and CDIR3 (minimum 9 %) in the present results probably occurred because the ewes were in an early stage of follicular development at the time of removal. This is supported by the fact that low values were not observed in CDIR4. Use of low doses of eCG (200 IU) contributed to a wider interval for ewes in estrus (24 to 54 h). For instance, when applying 400 IU eCG estrus onset occurs from 24 to 40 h². This happens because use of this dose in short- or long-term estrus synchronization protocols reduces the ovulation interval, accelerates ewes in estrus and stimulates greater estradiol production in the follicles¹⁸.

The pregnancy rates in the present results (77 to 88 %) were higher than reported in a study of CIDR devices used up to six times in 6-d protocols³. In this case, the difference may be due to the use of Awassi breed ewes, in which pregnancy and fertility rates decrease after the third use of a CIDR device. This is attributed to the low ewes in estrus rates in this study, and the use of fixed-time artificial insemination (FTAI)³. The present rates were also higher than the 70.1 % reported in a study of CIDR devices (new, second-use and third-use; no difference between treatments) in six-day protocols associated with 250 IU eCG and 0.133 mg cloprostenol sodium administered at removal¹². This discrepancy may be due partially to the use of natural mounting and mating for five days in an extensive system. In the present study, the ewes remained penned and were mated three times at a 12-h interval, which increased the probability of oocyte fertilization. The average prolificacy index in the present results was 1.26, which coincides with reported prolificacy values in Dorset and Katahdin ewes¹⁹.

Blood P₄ concentrations prior to device insertion indicate the ewes had begun their reproductive season. After insertion, concentrations increased in response to the supply of exogenous P₄ and remained above 1 ng/ml until device removal, which stimulated release of GnRH from the hypothalamus. Preovulatory increases in LH and FSH caused follicular growth and subsequent ovulation²⁰. This is deduced from the decrease of P₄ to 0.7 ng/ml at 72 h post device removal. Apparently, concentrations near 1 ng/ml did not negatively affect estrus synchronization since only 5% of the ewes did not exhibit estrus within 72 h post device removal.

Progesterone (P₄) is fundamental to maintaining early pregnancy and establishing implantation²⁰. Monitoring P₄ concentrations can therefore be useful in confirming pregnancy in ewes. The corpus luteum (CL) increases P₄ production on day three of pregnancy, increasing concentrations in maternal plasma to approximately 4 ng/ml by day seven²¹; this corresponds to the concentrations measured beginning on d8 in all four treatments of the present study. Increases in P₄ during this period are important because in sheep the CL is sensitive to the luteolytic action of PGF₂α between days four and fourteen of the estrous cycle²², and levels <1 ng/ml at this stage would not maintain a pregnancy. Remarkably, in the present results is that at the final sampling (d15) average P₄ concentration was lowest in CIDR1 (5.43 ng/ml), which did not differ from CIDR4. Higher levels can be linked to the higher CL number and total weight in sheep administered hormonal treatments²³; however, the differences in P₄ concentrations at the final sampling were not related to pregnancy rate or parturition type.

The use of CIDR devices in up to four six-day protocols with natural mating did not cause variation in reproductive variables between the four treatments. Progesterone (P₄) concentrations did not differ during the first three uses but did decline somewhat in the fourth. This highlights the potential of this estrus synchronization program since it can generate synchronization in a relatively short period. An added advantage of using CIDR devices in short periods (six days) is that it reduces ewe hormone exposure without negatively affecting reproductive variables (ewes in estrus, pregnancy and fertility rates, and prolificacy index). Clearly, CIDR intravaginal devices can be reused, although, because the devices are designed to release P₄ constantly, their effectiveness in estrus synchronization will depend on number of uses, sheep body weight and season (anestrus or reproductive). A final note is that reuse must occur within the same flock to avoid transmission of sexually-transmitted diseases between breeding animals.

Conclusions and implications

Use of six-day protocols allowed CIDR device reuse up to four times (i.e. new + three reuses) since they continue to release sufficient progesterone to block ovulation and synchronize estrus as effectively as new devices. The ewes in estrus, pregnancy and fertility rates did not decrease, while estrus onset interval, double births and the prolificity index were unaffected.