The work took place on the farm and in the Physiology laboratory of the Agricultural Research Institute for Development (IRAI) in Bambui (North-West Cameroon), from mid-March to June 2024. IRAD is located in 14 km from the town of Bamenda, in the district of Tubah, department of Mezam North-West Cameroon. This area is located at 05058' North Latitude and 10015' East Longitude and culminates at an average altitude of 1750 m. The vegetation consists of wooded savannah on the heights and gallery forest in the lowlands. In this region, 77% of the population is engaged in agriculture and livestock breeding with a cattle herd of approximately 56,038 heads [1] .

The selection of cows was done first on the basis of parity and age which were determined from the farm’s breeding records and the body condition score which was obtained by direct observation and interpretation following the grid of Vall et al. [12] .

An examination by palpation of the genital tract was also carried out on the selected cows, in order to ensure their physiological status (pregnant, empty or cycled). These operations made it possible to isolate 39 empty cows. Which were identified using ear tags. None of these females were lactating at the time of selection. The cows were fed on cultivated pasture (composed of Brachiaria ruziziensis and Penicetum Purpureum) and received dietary Supplementation (composed of maize, soya bean meal and CMAV 5%) once a week.

These females were those chosen according to our selection criteria (at least three months after the last birth, body condition score between 3 - 5, parity between 2 - 4 and non-lactating). In terms of health, they were free from tuberculosis and brucellosis, and vaccinated against major epizootic diseases (contagious bovine pleuropneumonia, foot-and-mouth disease).

These cows thus selected were divided into two groups, each subject to a heat induction/synchronization protocol. Each group was subsequently divided into two batches including a batch of adult cows (4 - 6 years old) and another batch of heifers (2 - 3 years old). The inseminations were carried out with fresh Holstein bull semen collected within the Bambui IRAD.

The 39 selected cows were divided into two groups:

The semen used for artificial inseminations was collected through a vagina artificial in a Holstein bull at IRAD Bambui. The collected semen was diluted with citrate and egg yolk, packaged in a 0.25 ml dose and refrigerated at 40˚C. The characteristics of the sperm were as follows:

Two heat synchronization protocols were each applied to a single group.

All artificial inseminations were carried out on average 48 + 2 hours after removal of the vaginal coils following the established protocol. Insemination was carried out using the recto-vaginal method and the deposition of the semen was uterine in cows with an open cervix and cervical in those with a closed cervix. The operation was carried out in such a way as to avoid any trauma and infection of the cows’ genital tract.

To carry out the actual artificial insemination operation, the cows were led and restricted in a containment corridor. The straws containing the seed were left at room temperature for around ten seconds, protected from the sun. The preparation of the Insemination Gun was done by rubbing on the external body in order to warm it. These straws were introduced into the insemination gun through their end having a double cap. The other heat-sealed end was cut perpendicular to the scissors to ensure maximum sealing with the cap of the insemination sheath. The gun and insemination sheath were covered with a protective plastic sheath that was punctured when the gun was inserted into the cervix.

With a gloved hand, the cervix was grasped through the rectal wall. Crossing the pass was made easier by giving it lateral and vertical movements. Once the pass was crossed, the gun was guided towards one or the other horn. The seed was placed there and the gun slowly removed then disinfected and cleaned for new use. At the end of the insemination each group of cows was separated from the other and concentrate was administered to them.

Blood samples were taken from the tail vein in all cows. These samples were taken in the afternoon and on eight occasions. First throughout the induction/synchronization process and after artificial insemination.

Blood was collected using dry tubes (VacUcheck@) which were identified and transported to the animal physiology laboratory of the Institute of Agricultural Research for Development (IRAD) in Bambui. Blood samples were centrifuged at 3200 rpm for 10 minutes to obtain serum. Blue tips attached to an automatic micropipette (1000 ml) were used to collect the serum which was transferred into Eppendorf tubes, identified and stored at −20˚C in the freezer.

Quantitative progesterone assays were carried out by the solid phase immunoenzymatic method (ELISA) as described by the commercial ClinPro progesterone EIA international kit.

To do this, 8 dosages were carried out at different periods of the heat induction/synchronization process and after artificial insemination.

Day 1 which corresponds to the day of installation of the vaginal spirals, Day 3, Day 5, Day 7 which is the day of removal of the vaginal spirals, Day 9 corresponding to the day of insemination, then 15 days after the AI, and finally 21 days after the AI.

According to Bayemi et al. [13] , the concentrations obtained after dosing with the ClinPro kit International left as follows:

Non-pregnancy was therefore confirmed for progesterone values lower than 3 ng/ml and the cows were assumed to be pregnant for progesterone values greater than or equal to 3 ng/ml at the end of the dosages, i.e. 21 days after the artificial insemination.

The characteristics of oxidative stress were as follows:

Determination of synchronization and pregnancy rates

Synchronization rate = (Number of cows present at the end of induction/synchronization × 100)/number of cow present

$\text{Pregnancy rate =}\frac{\text{Number of pregnant cows after diagnostics}\times \text{100}}{\text{Number of cows present}}$

Results were expressed as mean ± standard deviation and comparisons between dependent variables were made by analysis of variance (ANOVA). The Pearson correlation coefficient made it possible to establish the relationships between the different parameters. SPSS 21.0 software (Statistical Package for Social Sciences) was used for the analyzes and the significance limit set at 5%.

1) Variation in synchronization rate, serum progesterone concentration and oxidative stress level depending on heat induction/synchronization protocols and age of cows

Table 1 summarizes the effects of heat induction protocols and cow age on synchronization rate, progesterone concentrations, Malondialdehyde and Superoxide dismutase activity.

(a, b) On the same line, the values assigned the same letter do not differ significantly (P > 0.05). The second values correspond to the biochemical parameters of cows which showed no signs of heat. (ρ) correlation coefficient between Malondialdehyde level and Superoxide dismutase activity.

At the end of the hormonal treatments of the first synchronization protocol (Cidirol D0 and D7), only one cow out of 19 showed no sign of heat in the hours preceding insemination. The synchronization rate recorded was 94.47%. With percentages of manifestation of heat signs of 100% in adults (n = 9) and 90% in heifers (n = 10) respectively.

Concerning the second protocol (Cidirol D0 and D9), we recorded a synchronization rate of 70% (n = 20), including 90% in adults (n = 10) and only 50% in heifers (n = 10).

Regardless of protocols, the overall synchronization rate was 82.05%. The cows subjected to protocol I reacted better (94.47%) than those of the second (70%). For each Protocol, we also notice that the adults all reacted better than the heifers. With the heifers of the first protocol in which we recorded a rate of 90% (n = 10) compared to only 50% in the heifers of the second protocol (n = 10).

From the table, it appears that independently of the protocols, the levels of Progesterone, Malondialdehyde and the activity of Superoxide dismutase respectively do not differ significantly between the cows which showed apparent signs of heat and those which did not show any signs of heat.

The percentages of manifestation of heat signs were not significantly influenced by serum progesterone concentrations. However, progesterone concentrations were higher in cows that showed no signs of heat compared to cows in which these signs were observed.

Malondialdehyde concentrations and Superoxide dismutase activity respectively did not differ significantly between cows subjected to the two protocols.

No significant correlation was observed between Malondialdehyde levels and Superoxide dismutase activity. However, negative and non-significant correlations were recorded between progesterone levels and Superoxide dismutase activity respectively in adults of both protocols (ρ1 = −0.178; ρ2 = −0.850).

2) Variation in synchronization rate, progesterone profiles and oxidative stress depending on protocol type and breed

Table 2 presents the pregnancy rates, serum values of progesterone, Malondialdehyde concentration and Superoxide dismutase activity according to the protocols, and the breed of cows which showed signs of heat and those which did not show any signs.

(a, b) On the same line, the values assigned the same letter do not differ significantly (P > 0.05). The second values correspond to the biochemical parameters of cows which showed no signs of heat. (ρ) correlation coefficient between Malondialdehyde level and Superoxide dismutase activity).

For the first protocol (Cidirol D0 and D7), it appears that the Simmental breed cows all (100%) presented apparent signs of heat compared to the Borane breed cows (87.25) ( Table 2 ).

Concerning the second protocol (Cidirol D0 and D9), Goudali breed cows reacted better to treatments (100%) than Borane breed cows (40%).

The Borane and Simmental breed cows subjected to the first protocol reacted better than the Borane cows and the crossbreds of the second protocol.

Malondialdehyde level and Superoxide dismutase activity, respectively, did not differ significantly between cows of different breeds on the one hand between cows which showed signs of heat and those which did not react on the other hand.

No significant correlation was observed between Malondialdehyde levels and Superoxide dismutase activity.

3) Variation in synchronization rate, progesterone profiles and oxidative stress depending on protocol type and body condition score

The values of synchronization rates, serum progesterone concentration and oxidative stress according to the protocol type and body condition score of cows are reported in Table 3 .

(a, b) On the same line, the values assigned the same letter do not differ significantly (P > 0.05). The second values correspond to the biochemical parameters of the cows which showed no sign of heat. (ρ) Correlation coefficient between Malondialdehyde level and Superoxide dismutase activity.

Table 3 shows that the best synchronization rates were obtained in cows with a body condition score between 4 and 5: including 100% for the first protocol and 85% for the second.

As for the second protocol, cows with a body condition score (BCS) equal to 3.5 recorded a synchronization rate lower than that obtained in cows with an BCS between 4 - 5.

According to Table 3 , low concentration of progesterone was obtained in cows that showed signs of heat compared to cows that did not show. However, the significantly highest progesterone concentrations were observed in cows from the second protocol (Cidirol D0 and D9) of BCS 3.5 which showed heat.

Malondialdehyde concentration and Superoxide dismutase activity did not differ significantly between cows that showed signs of heat and between cows in which no signs of heat were observed before Artificial insemination.

Negative and significant correlation was recorded between the levels of Progesterone and Malondialdehyde concentrations in cows of protocol I (Cidirol D0 and D7) of BCS = 3.5 (ρ = −0.812^*) and positive and significant correlation was recorded between Malondialdehyde level and Superoxide dismutase activity in that of protocol 2 (ρ = 0.580^*).

4) Variation in pregnancy rate after artificial insemination

Eight assays were carried out to establish the progesterone profiles of the cows during the synchronization protocols and after artificial inseminations. Confirmation of non-pregnancy was effective at the last dosage, 21 days after artificial insemination.

Table 4 represents the pregnancy rates and the average progesterone values according to the heat induction protocols and to the age of the cow present.

(a, b) On the same line, the values of progesrterone assigned the same letter do not differ significantly (P > 0.05). The second values of progesrterone correspond to those of cows which showed no sign of heat.

Overall, there were 27 pregnant cows out of 39 inseminated, representing a pregnancy rate of 69.23%. According to Table 4 , cows whose heat was induced from the first Protocol recorded pregnancy rates higher than the rates of those subjected to the Second protocol (Cidirol D9). Regardless of age, the progesterone concentrations of cows in protocol I were significant (p > 0.05) higher than those in the second protocol.

Pregnancy rates after insemination and progesterone values according to heat induction protocols and breed are reported in Table 5 .

(a, b) On the same line, the values assigned the same letter do not differ significantly (P > 0.05). The second progesterone values correspond to empty cows.

Pregnant Borane cows and crossbreeds (protocol 2) recorded significantly lower levels of Progesterone than Goudali cows and those in the first protocol.

The pregnancy rate recorded in Borane cows subjected to the first Synchronization protocol was higher than the pregnancy rate of Borane cows subjected to the second protocol.

Post-insemination pregnancy rates and progesterone values based on heat induction protocols and body condition score are shown in Table 6 .

(a, b) On the same line, the values assigned the same letter do not differ significantly (P > 0.05). The second progesterone values correspond to empty cows.

Regardless of the protocol, the best pregnancy rates were recorded in cows with a body condition score of between 4 and 5. The pregnancy rate of cows with a body condition score of 3.5 in the second protocol was lower than that of those of the first. In these cows with a body condition score equal to 3.5 from protocol 2, the progesterone level was significantly (P < 0.05) the lowest compared to those noted in the other cows. The progesterone levels of pregnant cows subjected to the first protocol differed significantly from those of cows in the second protocol.

According to Table 7 , Malondialdehyde levels and Superoxide dismutase activity did not differ significantly between pregnant and empty breeds regardless of heat induction protocol. However, in empty cows, Superoxide dismutase activity is higher in empty cows compared to pregnant cows.

(ρ) correlation coefficient between Malondialdehyde levels and Superoxide dismutase activity. (^*) Significant at 0.05.

It appears from Table 7 that there is a positive and significant correlation between Malondialdehyde concentrations and Superoxide dismutase activity in Simmental cows (ρ = 664).

Table 8 also presents Malondialdehyde values and Superoxide dismutase activity according to protocols, cow body condition score and physiological status after AI.

(^*) Significant at 0.05; (^**) significant at 0.01.

Malondialdehyde levels and Superoxide Dismutase activity respectively did not differ significantly between pregnant and empty cows.

This table also reveals positive and significant correlations between Malondialdehyde concentrations and Superoxide dismutase activity in cows with body condition scores between 4 - 5 from both protocols (ρ1 = 0.938; P < 0.01, ρ2 = 0.530; P < 0.05).

Heat induction/synchronization protocols based on the use of progesterone are increasingly being developed in cattle. Some studies [16] - [18] ; report that the application of these intravaginal progesterone-releasing devices frequently causes oxidative stress which influences the fertility of animals undergoing artificial insemination. The present study evaluates the implication of certain factors linked to the animal in the oxidative stress induces by Heat induction/synchronization protocols.

After using two CIDR-based protocols, the synchronization rate obtained from the first synchronization protocol (94.47%) was higher than that obtained with the second (70%). This could be justified by the fact that the injection of Cidirol on the day of insemination in the cows of the second group did not have the expected effect (promoting the onset of heat). Especially since Cidirol is a product widely used in the heat synchronization protocol in cattle and containing a synthetic estradiol which promotes the onset of heat and ovulation. This late administration of Cidirol in the cows of the second Protocol would have contributed to its inferiority which would result in a weak manifestation of signs of heat in the cows of this protocol. The synchronization rate of cows in protocol 1 is quite similar to the rate of 98.04% reported by Seydou et al. [19] on Goudali cows with the same method, but lower than the 100% rate obtained by Abonou [16] in with the CIDR method on crosses. The synchronization rate obtained is also quite close to the rate of 66.66% observed by Vounparet et al. [17] on Arabian zebus with the CRESTAR method which is quite similar to the type of protocol applied to animals in the second group. The results also show that adults in these two protocols (100 and 90%) had better synchronization rates compared to heifers (90 and 50%). This could be explained by the fact that fertility increases in cattle with age and would be quite average in heifers in which the reproductive processors are still established.

It is known that better-fed cows show signs of heat better than cows whose diet is poor. Better synchronization rate was obtained in cows with Body Condition Scores between (BCS) 4 and 5. This could also be explained by the fact that reproductive performance in cattle increases with energy balance and the body condition of females and therefore, cows with better Body Condition Scores would have better fertility. Regardless of the protocols, 90% of the cows of BCS equal 3.5 which did not show signs of heat would be for the majority heifers.

In each group, correlations MDA levels were not significantly elevated. This would have favored the expression of quantities of progesterone coming from the spirals. However, no significant effect was observed on synchronization rate. It is still known that MDA has an effect on the growth process, maturation of follicles but also steroidogenesis and hence the appearance of heat. Also, the activity of MDA is known to increase the production of free radicals. It is also assumed that it is possible that the increase in MDA levels is local (at the ovarian level) and cannot be significantly detected at the serum level, due to the regulatory activity of SOD.

This observation noted in the present study differs from that of Kuru et al. [18] who noted that the quantity of MDA increased significantly after the removal of the spirals until the day of observation of heat, due to significant oxidative activity occurring during the installation and removal of the spirals.

Progesterone profiles were highlighted in cows from both groups. In non-pregnant females, the quantities of progesterone were high on the days following the placement of the coils (5 ng/ml), and reduced the day of estrus for cows in heat (unlike cows which did not show no sign of heat). Despite their decrease, progesterone concentrations remained high two weeks after insemination and reduced at the third week after insemination (<2 ng/ml). These results agree with those of Bayémi et al. [13] who revealed that the concentration of progesterone is low on the day of estrus, rises until the tenth day post-insemination and falls around the 21st day after insemination in females diagnosed as non-pregnant. Presumably pregnant females had progesterone concentrations that increased at the second and third week after AI (10 ng/ml). According to Thimonier [20] , the concentration of progesterone remains high throughout gestation. The application of pregnancy diagnosis by measuring progesterone levels in cows after artificial insemination is an excellent means of monitoring the success of this biotechnology. It is a tool widely used around the world, which allows the optimization of cattle production (helps reduce calving-to-calving intervals). During gestation, the concentration of progesterone remains high. This observation is the basis of this pregnancy diagnosis [21] .

Regardless of protocols, the overall pregnancy rate was 69.23%. 27 cows out of 39 were diagnosed as pregnant at the end of the Progeterone dosages. The cows subjected to protocol 1 recorded a pregnancy rate of 89.47% unlike the cows subjected to the second in which we noted a pregnancy rate of only 50%. The pregnancy rate of cows from the first protocol was slightly higher than 77.8% reported by Solihati [22] in dairy cattle FH using intravaginal progesterone with estrogen and injection 15 mg PGF2α intramuscularly and 80% obtained by Siregar et al. [23] with the CIDR-PGF2α method on the Aceh cow.

The pregnancy rate recorded by the cows in the second protocol was below the pregnancy rates recommended in AI (60% - 70%). It is also lower than the rate of 60% reported by Nakrani et al. [24] with the CRESTAR method in Holstein cows. Cows in protocol I responded significantly better to AI than those in the second. This is justified by the high rate of heat induction and synchronization caused by the first protocol compared to the second. This would have favored fertility, followed by the better pregnancy rate of group I cows. elsewhere, the low pregnancy rate recorded in group II cows could resulted from both oxidative stresses induce by insemination and injection of Cidirol at the same day.

Adults in both heat synchronization protocols (100% and 60%) obtained better pregnancy rates compared to heifers (80% and 40%). With a lower pregnancy rate recorded in heifers in the second group. This is explained firstly by the difference between the induction and heat synchronization protocols followed by the cows in the second group, because a poor luteal phase can influence the fertility of the cows and disadvantage the implantation and development of the conceptus. This observation could also be explained by the fact that fertility and fecundity increase with the increase in the number of births in cows and then reduce when the cows have reached more than 4 births. These results are in agreement with those of Tada et al. [25] who report a better pregnancy rate with cows aged between 4 - 6 years.

The lowest pregnancy rate was recorded in Borane and Goudali cows subjected to the second synchronization protocol. This could be justified by the fact that local cows generally have lower fertility than imported breed cows.

The best pregnancy rates were observed in cows which had BCS of 4 and 5. This result is closed to that reported by Dickinson et al. [26] according to whom the best rates are obtained in cows with a BCS of 4. Issa et al. [27] note that the body condition of cows at the time of insemination has a decisive influence on the pregnancy rate and should be monitored at each stage of reproduction. The best pregnancy rates noted in cows which BCS of 4 and 5 could be justify by appropriate proportion nutrients and good functioning of reproductive organs, necessary for gamete production, fecundation and gestation.

According to Celi et al. [28] , the oxidative stress attested by the imbalance between MDA level and SOD activity would be the cause of reproductive failures and cases of early embryonic mortality after AI. Serum MDA level and SOD activity were higher in empty Simmental and Goudali cows than in pregnant. This could be justified by the fact that MDA activities were more pronounced during the days preceding estrus in these cows, justifying the high level of oxidative stress responsible for degrading pregnancy. The MDA level and SOD activity were found to be higher in cows with the BCS of 3.5. According to Amel et al. [29] , oxidative activity would be more pronounced during energy imbalance and notes that oxidative stress would increase with the reduction in body condition score, due to the physiological processes that take place in order to compensate for energy deficits.