Follicle and corpus luteum size and vascularity as predictors of fertility at the time of artificial insemination and embryo transfer in beef cattle

Tamanho e vascularização do folículo e corpo lúteo como preditores de fertilidade após inseminação artificial e transferência de embriões em gado de corte

Abstract:

Two ultrasound based fertility prediction methods were tested prior to embryo transfer (ET) and artificial insemination (AI) in cattle. Female bovines were submitted to estrous synchronization prior to ET and AI. Animals were scanned immediately before ET and AI procedure to target follicle and corpus luteum (CL) size and vascularity. In addition, inseminated animals were also scanned eleven days after insemination to target CL size and vascularity. All data was compared with fertility by using gestational diagnosis 35 days after ovulation. Prior to ET, CL vascularity showed a positive correlation with fertility, and no pregnancy occurred in animals with less than 40% of CL vascularity. Prior to AI and also eleven days after AI, no relationship with fertility was seen in all parameters analyzed (follicle and CL size and vascularity), and contrary, cows with CL vascularity greater than 70% exhibit lower fertility. In inseminated animals, follicle size and vascularity was positive related with CL size and vascularity, as shown by the presence of greater CL size and vascularity originated from follicle with also greater size and vascularity. This is the first time that ultrasound based fertility prediction methods were tested prior to ET and AI and showed an application in ET, but not in AI programs. Further studies are needed including hormone profile evaluation to improve conclusion.

Index Terms:
Fertility; follicle; corpus luteum; ultrasound; color-Doppler; cattle

Resumo:

Duas técnicas de predição de fertilidade, baseadas em ultrassonografia, foram testadas no momento da transferência de embriões (TE) e inseminação artificial (IA) em bovinos. Fêmeas bovinas foram submetidas a protocolos de sincronização de estro para TE e IA. Os animais foram escaneados por ultrassonografia imediatamente antes do procedimento de TE e IA para identificar o tamanho e vascularização do folículo e corpo lúteo (CL). Além disso, os animais inseminados foram escaneados onze dias após a inseminação para identificar o tamanho e vascularização do CL. Todos os dados foram comparados com a fertilidade utilizando-se do diagnóstico gestacional 35 dias após a ovulação. No momento da TE, a vascularização do CL apresentou-se positivamente relacionada com a fertilidade, sendo que animais com menos de 40% de vascularização do CL não ficaram gestantes. No momento da IA, assim como onze dias após a IA, nenhuma relação foi encontrada entre fertilidade e os parâmetros analizados (tamanho e vascularização do folículo e CL), enquanto que contrariamente, houve uma queda na fertilidade em vacas com vascularização do CL acima de 70%. Nos animais inseminados, o tamanho e vascularização do folículo foi positivamente relacionado ao tamanho e vascularização do CL, demonstrado pela presença de um CL maior e mais vascularizado proveniente de um folículo maior e mais vascularizado. O presente estudo é o primeiro a mostrar métodos de predição de fertilidade baseados em ultrassonografia no momento da IA e TE, demonstrando uma aplicabilidade prática no momento da TE. Novos estudos são necessários para suportar os resultados mostrados, incluindo análises hormonais.

Termos de Indexação:
Fertilidade; folículo; corpo lúteo; ultrassonografia; Doppler colorido; bovino

Introduction

Assisted reproduction techniques (ART) are highly diffused in cattle industry, aiming to increase herd fertility as well synchronize births and homogenate the herd age for slaughter. Artificial insemination (AI) and embryo transfer (ET) can be defined as the major ART techniques in cattle industry. These two ART techniques are efficient and with suitable results for cattle production, although a "fine tuning" is necessary to achieve even greater reproductive results.

New studies aimed to increase herd fertility index by hormone treatments (Bisinotto et al. 2011) or improving semen quality (Kastelic et al. 2008). However, the evaluation of the success of each new approach used to increase herd fertility is, in practice, only measured by gestational diagnosis 35 days after ovulation. Earlier pregnancy detection is not been used yet, although was already tested (Siqueira et al. 2013), mainly by transrectal ultrasonography. Early gestational diagnosis had aimed to improve herd production, since it could enable an early reintroduction of the non-pregnant cows in reproduction, and an increased number of pregnant animals in a shorter time. In addition, real time methods to predict fertility at the time of AI and ET procedure were also previously tested (Silva et al. 2006, Siddiqui et al. 2008, Sá Filho et al. 2010a). Therefore, an immediate diagnosis to help the decision of using or discarding an animal from an AI or ET program is still questionable.

Follicle vascularity and its effects on fertility had been investigated in mares (Silva et al. 2006) and cows (Shrestha et al. 2006, Siddiqui et al. 2008). Near ovulation, was shown that an extensive vascularity is formed in the follicular wall until the ovulation occurs (Geva & Jaffe 2000, Reisinger et al. 2007). Subjective and objective methods are validated and can be used to measure follicular vascularity in cows and mares (Ginther et al. 2007). Previous works showed that follicles with greater diameter resulted in also greater pregnancy rates (Sá Filho et al. 2010a). Furthermore, preovulatory follicles with greater size originated CLs with greater size and presenting higher production of progesterone (Bisinotto et al. 2012, Machado et al. 2008).

Several studies addressed the role of the vascularity of genital organs in the estrous cycle, reproductive life, and gestation (Reed et al. 1996, Nautrup 1998, Bollwein et al. 2000, 2002a, 2002b, 2004, Köster et al. 2001, Alvarez-Clau & Liste 2005, Di Salvo et al. 2006, Scotti et al. 2008, Brito et al. 2010, Miranda & Domingues 2010, Polisca et al. 2010, Blanco et al. 2011, Pereira et al. 2012a, 2012b). Color-Doppler ultrasonography was previously used in cows to evaluate the vascularity of follicle, CL, and conceptus (Miyamoto et al. 2005, 2006, Pareja et al. 2010, Silva et al. 2010). Therefore, since it is a non invasive technique and with reliable application, it is a useful tool to target new reproductive diagnoses. The evaluation of perifollicular vascularity was done in cows (Siddiqui et al. 2008), women (Coulam et al. 1999, Bhal et al. 2001, Borini et al. 2001), and mares (Silva et al. 2006), and in all of these species was detected greater vascularity in the preovulatory follicle wall in individuals that became pregnant after mating.

Ultrasonography in B- and color-Doppler modes of the CL can potentially be used as a diagnosis tool to select better recipients for an ET program, as well to early detect gestational losses. Previously, it was noted that pregnancy rate was not correlated with CL diameter (Nunes et al. 2011), although greater CL area was positive correlated with pregnancy (Baruselli et al. 2003, Nunes et al. 2011). Measurement of CL vascularity was suggested as a diagnosis technique for early pregnancy, but the early detection of pregnancy by evaluation of CL vascularity did not appeared to be a specific and sensitive method (Utt et al. 2009). The physiologic aspects of CL vascularity by ultrasonography during formation and lysis were previously described in cows (Acosta et al. 2003, Acosta & Miyamoto 2004, Miyamoto et al. 2005, Ginther et al. 2007), using objective and subjective analyses (Ginther et al. 2007). The corpus luteum vascularity is immediately constituted after ovulation (Reynolds et al. 2000), and CL vascularity is associated with its functionality (Bollwein et al. 2002b). CL size and vascularity is positively correlated with progesterone production, and appeared to be a suitable predictor of progesterone secretion (Acosta et al. 2003, Mann 2009). In addition, the evaluation of CL vascularity should be a useful method to evaluate conceptus development, since greater concentrations of progesterone in the uterine environment during early stages of gestation was related with improved embryonic development (Lonergan 2011).

Thereby, two experiments were proposed to test the use of color Doppler ultrasonography to detect the success of AI of ET. The following hypotheses were tested: 1) Corpus luteum with greater vasculature at the time of ET results in greater pregnancy rates after ET; 2) Larger pre-ovulatory follicles presenting greater vascularity generates larger corpus luteum also presenting greater vascularity, resulting in a greater pregnancy rates after AI.

Materials and Methods

Animals and ultrasound scanning. Nellore cows (n=198), Nellore primiparous (n=80), Nellore heifers (n=51), and Tabapuã cows (n=21) were used during November to February in the Southern Hemisphere Tropical Zone. Animals were selected with no abnormalities in the reproductive tract, as determined by ultrasound examinations (Ginther 1998). Animals were kept under natural light and had free access to Brachiria brizantha pastures, traced-mineralized salt and water. Handling of animals was in accordance with the Institutional Animal Care and Use Committee (CEUA-FMVZ/USP, No.3030/2013).

A duplex pulsed-wave color-Doppler ultrasound instrument (MyLab30 Vet Gold; Esaote Healthcare, Genova, Italy) equipped with a multifrequency linear transducer for large animals transretal exams was used for the scanning. For the gray-scale mode, the brightness and contrast controls of the monitor and the gain controls of the scanner were standardized to constant settings (Gastal et al. 2006). For color Doppler mode, all scans were performed at a constant color-gain setting and a velocity setting of 5cm/s (Pugliesi et al. 2014). All scans were recorded by video clips for further analyses using specific software (MyLab Desk; Esaote Healthcare, Genova, Italy).

Image analyses. Video clips of the scanning were used to select still frames of the preovulatory follicles (POF) and corpus luteum (CL). The maximum diameter of POF and CL was determined using the software caliper function, and the mean of two perpendicular diameters were used for analyzes. The maximum CL area was determined using the software tracing function. For CL with cavity, the diameter and area of the cavity was subtracted from the total area (Kastelic et al. 1990). The percentage of POF wall and luteal tissue with vascular signals was determined subjectively by watching the color Doppler video clip, as previously described (Ginther et al. 2007).

Experiment 1. Nellore cows (n=43) were submitted to the protocol of estrus synchronization as described in Figure 1. Seven days after ovulation, ultrasound scan was done searching for CL, and at the presence of CL the cow was submitted to embryo transfer. All scans were done by the same operator and a video clip recorded in B-mode and color-Doppler mode for further analyzes of CL diameter, area, and vascularity.

Fig.1:
Protocol of estrous synchronization for fixed-timed embryo transfer (Experiment 1).

The embryos were produced by in vitro fertilization, using oocytes from Brangus breed cows and semen from the same bull and all were graded as one for quality (International Embryo Transfer Society, IETS). The embryo transfer procedure was done by the same operator, and the embryo placed at the uterine horn ipsilateral of the ovary with CL. Gestational diagnosis was done thirty days after ovulation by ultrasound scanning. The experiment 1 design is shown in Figure 2.

Fig.2:
Experimental design (Experiment 1).

Experiment 2. Heifers, primiparous and pluriparous Nellore cows and pluriparous Tabapuã cows (total of animals = 307) were submitted to the same protocol of estrous synchronization for artificial insemination, as described in Figure 3. All animals were inseminated one day after ovulation using semen from the same bull. Ultrasound scans were done by the same operator at the time of insemination and eleven days after, to search for POF and CL, respectively. A video clip of the POF and CL scanning were recorded in B-mode and color-Doppler mode for further analyzes of POF diameter and vascularity; as well CL diameter, area and vascularity. The gestational diagnosis was done thirty days after ovulation by ultrasonography. Experiment 2 design is shown in Figure 4.

Fig.3:
Protocol of estrous synchronization for fixed-time artificial insemination (Experiment 2)

Fig.4:
Experimental design (Experiment 2).

Statistical analyses. Comparisons of proportions were done by chi-square. Objective data normality was verified by Kolmogorov-Smirnov test. Data not normally distributed were transformed in log of rank. Variance analyzes were done by SAS program (GLM procedure; version 9.2; SAS Institute Inc., Cary, NC) to determine principal effects and interaction. The student t-test was used to search differences between means in the same group or among groups if principal effects or interaction was significant. The probability between P≤0.10 and P>0.05 indicated approaching significance and P ≤ 0.05 indicated significance.

Results

Experiment 1

Cows were divided in two groups according to CL vascularity at the time of embryo transfer: G≤40 (0-40%), G>50 (41-100%). The means of CL diameter and pregnancy rate are shown in Table 1. A positive effect between CL vascularity and pregnancy rate was found, although no effect was found between CL size and pregnancy rate. In addition, the CL diameter was not correlated with vascularity.

Table 1:
Mean of CL diameter and pregnancy rate in the groups divided by percentage of vascularity of the CL (Experiment 1). a and b represents differences between treatments by chi-square test (P>0.05).

Experiment 2

Animals were divided in five groups according to CL vascularity 11 days after AI: G 40 (0-40%), G 50 (41-50%), G 60 (51-60%), G 70 (61-70%), and G 100 (70-100%). No difference was found between CL vascularity and pregnancy rate, except for the group G 100, in which pregnancy rate was lower. In addition, the CL diameter was not correlated with vascularity, as shown in Table 2.

Table 2:
Mean of CL diameter and pregnancy rate in the groups divided by percentage of vascularity of the CL (Experiment 2)

• P<0.05.

Data was arranged in ascending order for POF diameter, POF vascularity, CL diameter, CL area, CL vascularity, and CL area of vascularity. No effect was found between any of the parameters on pregnancy rate, although was found a tendency (P=0.07) effect of CL area on pregnancy rate (Table 3).

Table 3:
POF diameter and vascularity; CL diameter, area, vascularity, and area of vascularity of the animals with positive (n=155) and negative (n=151) gestational diagnosis (GD)

• No statistical differences (P<0.05) were found among parameters. Approaching to significance was found for CL area. Data were analyzed using unpaired student t-test.

For additional comparisons, data was divided in two groups of low values and high values from the median (Table 4). Thereby, POF diameter affected positively CL area, vascularity and area of vascularity. POF vascularity affected positively CL vascularity and area of vascularity. CL diameter affected positively CL area and vascularity. CL area affected positively POF diameter and vascularity, and CL diameter. CL vascularity affected positively POF diameter and vascularity, and CL area of vascularity. CL area of vascularity affected positively all parameters.

Table 4:
Data arranged in groups with greater and lower values from the median. Analyzed parameters: POF diameter and vascularity; CL diameter, vascularity, area, and area of vascularity

• Bold values represent the MEAN ± SEM of the parameter used to divide the groups. Values with significant differences are highlighted in grey. Superscript low case letters represent the following p values:
• ^a
  p=0.0004,
• ^b
  p=0.001,
• ^c
  p<0.0001,
• ^d
  p=0.0007,
• ^e
  p=0.02,
• ^f
  p<0.0001,
• ^g
  p<0.0001,
• ^h
  p=0.0001,
• ⁱ
  p<0.0001,
• ^j
  p<0.0001,
• ^k
  p<0.0001,
• ^l
  p=0.003,
• ^m
  p<0.0001,
• ⁿ
  p<0.0001,
• ^o
  p=0.006,
• ^p
  p<0.0001,
• ^q
  p<0.0001,
• ^r
  p<0.0001.

Data was also organized in quartiles, and two groups were formed based on the first and third quartile for each parameter (Table 5). POF diameter affected positively POF vascularity, CL area, CL vascularity, and CL area of vascularity. POF vascularity affected positively POF diameter, CL vascularity, and CL area of vascularity. CL diameter affected positively POF diameter, CL diameter, CL area, CL and vascularity. CL area affected positively POF diameter, CL diameter, and CL area of vascularity. CL vascularity affected positively POF diameter, POF vascularity, and CL area of vascularity. CL area of vascularity affected positively POF diameter, CL area, and CL area of vascularity.

Table 5:
Data divided groups composed by the first and third quartiles of the data. Analyzed parameters: POF diameter and vascularity; CL diameter, vascularity, area, and area of vascularity

• Bold values represent the MEAN±SEM of the parameter used to divide the groups. Values with significant differences are highlighted in grey. Superscript low case letters represent the following p values:
• ^a
  p=0.05,
• ^b
  p<0.0001,
• ^c
  p<0.0001,
• ^d
  p<0.0001,
• ^e
  p=0.007,
• ^f
  p=0.004,
• ^g
  p=0.03,
• ^h
  p<0.0001,
• ⁱ
  p<0.0001,
• ^j
  ^p<0.0001,
• ^k
  ^p<0.0001,
• ^l
  p<0.0001,
• ^m
  p<0.0001,
• ⁿ
  p<0.0001,
• ^o
  p=0.0004,
• ^p
  p<0.0001,
• ^q
  p<0.0001,
• ^r
  p<0.0001,
• ^s
  p<0.0001,
• ^t
  v<0.0001.

Discussion

Measurements of follicle and CL size and vascularity were done prior to artificial insemination and embryo transfer. Previous studies also assessed these parameters in cattle (Siddiqui et al. 2008.), horses (Silva et al. 2006), and humans (Coulam et al. 1999, Bhal et al. 2001, Borini et al. 2001), and encountered a positive correlation between FL vascularity and fertility. In the present study, CL vascularity was positive related with pregnancy rate at the time of embryo transfer, but no relationship with fertility was noted when follicle size and vascularity were measured at the time of AI.

A positive correlation with follicle size at the time of AI and pregnancy rate was previously described (Sá Filho et al 2010a). Follicle and CL size were also positive related (Wecker et al. 2012), but no interference in fertility was noted. In the present study, no relationship between CL size with fertility was noted, similarly as observed by Nuñes et al. (2011), although disagreeing with findings from Baruselli et al. (2003), Baruselli et al. (2004)and Sá Filho et al. (2010a, 2010b, 2010c).

The hypothesis 1 .... that corpus luteum with greater vasculature at the time of ET results in greater pregnancy rates after ET .... was supported. In experiment 1, pregnancy rate was greater in cows with greater vascularity of the CL. This finding can be supported by Bollwein et al. (2002b) data, in which was noted greater CL vascularity associated with higher CL function. Previous studies indicated that CL vascularity may represent fertility and, since CL vascularity increases in parallel with systemic circulation of progesterone, it may be a useful method for pregnancy diagnosis (Acosta et al. 2003, Utt et al. 2009). In addition, higher CL function is also associated with greater progesterone concentration in the uterine environment, which is associated with improved embryo development (Lonergan 2011). The sum of these characteristics of CL vascularity supports that the positive correlation between CL vascularity and fertility may be due to increased systemic progesterone concentration. However, in the present study systemic progesterone was not measured, and further studies are necessary.

The hypothesis 2 that pre-ovulatory follicles larger and with greater vasculature generates larger and with greater vasculature corpus luteum, resulting in a greater pregnancy rates after AI was partially supported. In experiment 2, greater follicular size and vascularity originated also greater CL size and vascularity. This finding may be a result of the increased systemic estradiol, as shown previously (Reynolds et al. 2000, Bollwein et al. 2002a). Although a positive correlation among follicular size, follicular vascularity, CL size, and CL vascularity was observed, no benefit on fertility among each of these parameters was noted, and in contrast lower fertility rate was noted in cows with CL vascularity greater than 70%. Hypothetically, the animals used in experiment 2 were maintained in over nutrition and were extremely selected for fertility, which could be the reason for no significant differences on pregnancy rate among all parameters analyzed. A herd with possible greater variability of reproductive status (different degrees of body scores or inseminated in two different climate seasons), should be used for further investigation, and test the applicability of the evaluation of follicle size and vascularity in practice. In addition, the lower fertility rate in cows with the greatest CL vascularity was an unexpected finding and with weak support owing to the low number of animal. However, previous studies (Parr et al. 2012) showed a deleterious effect of higher concentration of progesterone in the embryo viability, and appeared to be a suitable hypothesis for the low fertility rate in these animals with higher CL vascularity, which indirectly showed higher CL function and, probably, a higher progesterone concentration in the blood.

In conclusion, in the present study fertility was positively related with CL vascularity prior to embryo transfer procedure, but no relationship between all parameters analyzed in follicle were associated with fertility prior to artificial insemination. The conflicting results after AI and ET is suggested to be a consequence from different herds conditions and needs to be tested. Therefore, further studies including more heterogeneous herds and hormone profiles are necessary to improve these findings.

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Publication Dates

• Publication in this collection
  May 2015

History

• Received
  23 June 2014
• Accepted
  13 Dec 2014